Antibodies to Non-Functional Oligomeric P2X7 Receptors

ABSTRACT

The invention relates to purinergic receptors, to antibodies and related fragments thereof for binding to said receptors, to production of said antibodies and fragments and to use of said antibodies and fragments for cancer detection and therapy. In particular the antibodies described bind specifically to non-functional P2X7 receptors expressed by live cells.

FIELD OF THE INVENTION

The invention relates to purinergic receptors, to antibodies and relatedfragments thereof for binding to said receptors, to production of saidantibodies and fragments and to use of said antibodies and fragments forcancer detection and therapy.

BACKGROUND OF THE INVENTION

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction or that this prior art could reasonably be expected to beascertained, understood and regarded as relevant by a person skilled inthe art.

Purinergic (P2X) receptors are ATP-gated cation-selective channels. Eachreceptor is made up of three protein subunits or monomers. To date sevenseparate genes encoding P2X monomers have been identified: P2X₁, P2X₂,P2X₃, P2X₄, P2X₅, P2X₆, P2X₇.

P2X₇ receptors are of particular interest as the expression of thesereceptors is understood to be limited to cells having potential toundergo programmed cell death, such as thymocytes, dendritic cells,lymphocytes, macrophages and monocytes. There is some expression of P2X₇receptors in normal homeostasis, such as on erythrocytes.

Interestingly, a P2X₇ receptor containing one or more monomers having acis isomerisation at Pro210 (according to SEQ ID NO: 1) and which isdevoid of ATP binding function has been found on cells that areunderstood to be unable to undergo programmed cell death, such aspreneoplastic cells and neoplastic cells. This isoform of the receptorhas been referred to as a “non functional” receptor.

Antibodies generated from immunisation with a peptide including Pro210in cis bind to non functional P2X₇ receptors. However, they do not bindto P2X₇ receptors capable of binding ATP. Accordingly, these antibodiesare useful for selectively detecting many forms of carcinoma andhaemopoietic cancers and to treatment of some of these conditions.

WO02/057306A1 and WO03/020762A1 both discuss a probe for distinguishingbetween functional P2X₇ receptors and non functional P2X₇ receptors inthe form of a monoclonal antibody.

To date it has been very difficult to obtain serological reagents thatbind to non functional P2X₇ receptors on live cells with desirableaffinity. Higher affinity reagents are generally desirable inapplications for the detection and treatment of cancer.

There is a need for improved reagents for binding to P2X₇ receptors,particularly for new antibodies and fragments thereof that are capableof discriminating between ATP and non-ATP binding P2X₇ receptors on livecells. There is also a need for antibodies and fragments thereof thatexhibit preferential binding to a P2X₇ receptor as it is expressed onlive cells with reduced capacity to bind to a P2X₇ receptor once thetarget cell has died.

SUMMARY OF THE INVENTION

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 1:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: (charged/polar/aromatic)(charged/aromatic)XXXY(aromatic/aliphatic)(charged/neutral)(neutral/aliphatic).X throughout the specification represents any amino acid.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 2:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: N(Y/F)XXXY(Y/F)EX.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 3:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of:N(Y/F)(neutral)(charged)(neutral)Y(Y/F)E(neutral).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 4:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: NFLESYFEA.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 5:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of:N(Y/F)(charged)(neutral)(charged)Y(Y/F)E(neutral).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 6:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: NYRGDYYET.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 7:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: H(aromatic)XXXYYNI.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 8:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of:H(Y/F)(neutral)(charged)(charged)YYNI.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 9:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of:H(Y/F)(neutral)(charged)(neutral)YYNI.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 10:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: HYSKEYYNI.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 11:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: HFQRGYYNI.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 12:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of:(Y/N)(aromatic))XXXYY(charged)(neutral).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 13:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of:(Y/N)(aromatic)(neutral)(neutral)(neutral)YYDV.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 14:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of:(Y/N)(aromatic)(neutral)(neutral)(neutral)YYEV.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 15:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: YFPLVYYDV.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 16:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: NYLPMYYEV.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 17:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of:Y(charged)XXXY(neutral)(neutral)(neutral).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 18:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: YHVIQYLGP.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 19:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence selected from the group consisting of:

HYSSRFFDV, NFKLMYYNV, NYRGDYYET, HFSRGYYDV, NFLESYFEA,NYLPMYYEV, HYIKVYYEA, HYSSRFFEV, NFRVMFFKA, HFQRGYYNI, HYSSRFFEV,YHVIQYLGP, HYSKEYYNI, YFPLVYYDV, DFTVPFYNA, NYDKKYFDV, YFPLVYYDV.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 20:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has an amino acid sequence of KASQNVGTNVA.CDR3 has an amino acid sequence of any previous embodiment describing aCDR3 sequence.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 21:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has an amino acid sequence of SYYMS.CDR3 has an amino acid sequence of any previous embodiment describing aCDR3 sequence.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 22:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR2 has an amino acid sequence of SASFRYS.CDR3 has an amino acid sequence of any previous embodiment describing aCDR3 sequence.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 23:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR2 has an amino acid sequence of AINSNGGSTYYPDTVKG.CDR3 has an amino acid sequence of any previous embodiment describing aCDR3 sequence.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 24:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has an amino acid sequence of KASQNVGTNVACDR2 has an amino acid sequence of SASFRYSCDR3 has an amino acid sequence of any previous embodiment describing aCDR3 sequence.

In one embodiment there is provided an antigen binding site for binding,to a P2X₇ receptor, the antigen binding site being defined by generalformula 25:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR1 has an amino acid sequence of SYYMSCDR2 has an amino acid sequence of AINSNGGSTYYPDTVKGCDR3 has an amino acid sequence of any previous embodiment describing aCDR3 sequence.

In one embodiment there is provided an antigen binding site according toany embodiment described above wherein FR1 is eitherMADIVMTQSQKFMSTSVGDRVSVTC or DVKLVESGGGLVKLGGSLKLSCAASGFTFS.

In one embodiment there is provided an antigen binding site according toany embodiment described above wherein FR2 is either WYQQKPGQSPKALIY orWVRQTPEKRLELVA.

In one embodiment there is provided an antigen binding site according toany embodiment described above wherein FR3 is eitherGVPDRFTGSGSGTDFTLTISNVQSEDLAEFFC Or RFTISRDNAKNTLYLQMSSLKSEDTAFYYCTR.

In one embodiment there is provided an antigen binding site according toany embodiment described above wherein FR4 is either FGSGTRLEIK orWGAGTTVTVSS.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 26:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a

wherein:FR1, FR2, FR3, FR4, FR1a, FR2a, FR3a and FR4a are each frameworkregions;CDR1, CDR2, CDR3, CDR1a, CDR2a, CDR3a are each complementaritydetermining regions;wherein:CDR1 has an amino acid sequence of KASQNVGTNVACDR2 has an amino acid sequence of SASFRYSCDR3 has an amino acid sequence of any previous embodiment describing aCDR3 sequence or QQYNSYPFT.CDR1a has an amino acid sequence of SYYMSCDR2a has an amino acid sequence of AINSNGGSTYYPDTVKGCDR3a has an amino acid sequence of any previous embodiment describing aCDR3 sequence or QQYNSYPFT (SEQ ID NO: 33) when CDR3 is an amino acidsequence of any previous embodiment describing a CDR3 sequenceFR1 has an amino acid sequence of MADIVMTQSQKFMSTSVGDRVSVTC (SEQ ID NO:25)FR2 has an amino acid sequence of WYQQKPGQSPKALIY (SEQ ID NO: 26)FR3 has an amino acid sequence of GVPDRFTGSGSGTDFTLTISNVQSEDLAEFFC (SEQID NO: 27)FR4 has an amino acid sequence of FGSGTRLEIK (SEQ ID NO: 28)FR1a has an amino acid sequence of DVKLVESGGGLVKLGGSLKLSCAASGFTFS (SEQID NO: 29)FR2a has an amino acid sequence of WVRQTPEKRLELVA (SEQ ID NO: 30)FR3a has an amino acid sequence of RFTISRDNAKNTLYLQMSSLKSEDTAFYYCTR (SEQID NO: 31)FR4a has an amino acid sequence of WGAGTTVTVSS (SEQ ID NO: 32).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 27:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of(charged/polar/aromatic)(aromatic)(charged/neutral)(charged)(charged/neutral)Y(aromatic)(charged)(neutral).

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 28:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of:(charged/polar/aromatic)(F/Y)(charged/neutral)(R/K)(charged/neutral)(Y)(Y/F)(E/D)V.

In one embodiment there is provided an antigen binding site for bindingto a P2X₇ receptor, the antigen binding site being defined by generalformula 30:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein:FR1, FR2, FR3 and FR4 are each framework regions;CDR1, CDR2 and CDR3 are each complementarity determining regions;wherein:CDR3 has an amino acid sequence of: (H/N)(F/Y)(S/D)(R/K)(G/K)Y(Y/F)DV.

In one embodiment the linker of general formula 26 has an amino acidsequence of 15 amino acid residues. Typically, the linker comprisespredominately glycine and serine residues. Preferably, the linker isGGGGSGGGGSGGGGS.

In one embodiment, the antigen binding site of the invention has a CDR3amino acid sequence that comprises HFSRGYYDV or NYDKKYFDV.

In one embodiment, the antigen binding site of the invention has a CDR3amino acid sequence that consists of HFSRGYYDV or NYDKKYFDV.

In other embodiments there is provided an antigen binding site having asequence as described herein, or including a CDR and/or FR sequence asdescribed herein and including one or more mutations for increasing theaffinity of said site for binding to a P2X₇ receptor.

In another embodiment there is provided an antigen binding site asdescribed herein wherein an amino acid sequence forming one or more ofFR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 is a human sequence.

In another embodiment there is provided an antigen binding site asdescribed herein wherein an amino acid sequence forming one or more ofFR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 is a canine or feline sequence.

The antigen binding site may be engineered to have sequences from aparticular animal, for example it may be chimeric (i.e. containing somebut not all sequences found in the individual that receives theantibody). Alternatively, it may consist of allogeneic or syngeneicsequences. An example of the latter is a dog antibody for use intreatment of a dog.

The animal from which the antibody is derived may include a domestic,companion or farm animal, including dogs, cats, cows, pigs, horses andsheep.

In another embodiment there is provided an anti P2X₇ receptorimmunoglobulin variable domain, antibody, Fab, dab, scFv including anantigen binding site having a sequence as described herein, or includinga CDR and/or FR sequence as described herein.

In another embodiment there is provided a diabody or triabody includingan antigen binding site having a sequence as described herein, orincluding a CDR and/or FR sequence as described herein.

In another embodiment there is provided a fusion protein including anantigen binding site, immunoglobulin variable domain, antibody, Fab,dab, scFv, diabody or triabody as described herein.

In another embodiment there is provided a conjugate in the form of anantigen binding site, immunoglobulin variable domain, antibody, Fab,dab, scFv, diabody, triabody or fusion protein as described hereinconjugated to a label or a cytotoxic agent.

In another embodiment there is provided an antibody for binding to anantigen binding site of an immunoglobulin variable domain, antibody,Fab, dab, scFv, diabody, triabody, fusion protein, or conjugate asdescribed herein.

In another embodiment there is provided a nucleic acid encoding anantigen binding site, or a CDR and/or FR sequence as described herein,or an immunoglobulin variable domain, antibody, Fab, dab, scFv, diabody,triabody, fusion protein or conjugate as described herein.

In another embodiment there is provided a vector including a nucleicacid described herein.

In another embodiment there is provided a cell including a vector ornucleic acid described herein.

In another embodiment there is provided an animal or tissue derivedtherefrom including a cell described herein.

In another embodiment there is provided a pharmaceutical compositionincluding an antigen binding site, or including a CDR and/or FR sequenceas described herein, or an immunoglobulin variable domain, antibody,Fab, dab, scFv, diabody, triabody, fusion protein, or conjugate asdescribed herein and a pharmaceutically acceptable carrier, diluent orexcipient.

In another embodiment there is provided a diagnostic compositionincluding an antigen binding site, or including a CDR and/or FR sequenceas described herein, or an immunoglobulin variable domain, antibody,Fab, dab, scFv, diabody, triabody, fusion protein or conjugate asdescribed herein, a diluent and optionally a label.

In another embodiment there is provided a kit or article of manufactureincluding an antigen binding site, or including a CDR and/or FR sequenceas described herein or an immunoglobulin variable domain, antibody, Fab,dab, scFv, diabody, triabody, fusion protein or conjugate as describedherein.

In another embodiment there is provided a use of a sequence according toone or more of CDR1, CDR2, FR1, FR2, FR3 and FR4 as described herein toproduce an antigen binding site for binding to a P2X₇ receptor.

In another embodiment there is provided a use of an antigen binding siteor a CDR and/or FR sequence as described herein to produce an anti P2X₇receptor antigen binding site having increased affinity for P2X₇receptor.

In another embodiment there is provided a library of nucleic acidmolecules produced from the mutation of an antigen binding site or a CDRand/or FR sequence as described herein, wherein at least one nucleicacid molecule in said library encodes an antigen binding site forbinding to an a P2X₇ receptor.

In another embodiment there is provided a method for producing an antiP2X₇ antigen binding site as described herein including expressing anucleic acid as described herein in a cell or animal as describedherein.

In another embodiment there is provided a method for the treatment ofcancer or a condition or disease associated with expression of nonfunctional P2X₇ receptor in an individual including the step ofproviding an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein, conjugateor pharmaceutical composition as described herein to an individualrequiring treatment for cancer or said condition or disease.

In another embodiment there is provided a use of an antigen bindingsite, immunoglobulin variable domain, antibody, Fab, dab, scFv, diabody,triabody, fusion protein, conjugate or pharmaceutical composition asdescribed herein in the manufacture of a medicament for the treatment ofcancer or a condition or disease associated with expression of nonfunctional P2X₇ receptor.

In another embodiment there is provided an antigen binding site,immunoglobulin variable domain, antibody, Fab, dab, scFv, diabody,triabody, fusion protein, conjugate or pharmaceutical composition asdescribed herein for the treatment of cancer or a condition or diseaseassociated with expression of non functional P2X₇ receptor.

In another embodiment there is provided a method for the diagnosis ofcancer or disease or condition associated with expression of nonfunctional P2X₇ receptor, including the step of contacting tissues orcells for which the presence or absence of cancer is to be determinedwith a reagent in the form of an antigen binding site, immunoglobulinvariable domain, antibody, Fab, dab, scFv, diabody, triabody, fusionprotein, conjugate or diagnostic composition as described herein anddetecting for the binding of the reagent with the tissues or cells. Themethod may be operated in vivo or in vitro.

Typically the antigen binding sites according to the invention bind tonon functional P2X₇ receptors, especially receptors wherein Pro210 ofP2X₇ is in cis conformation. In certain embodiments the antigen bindingsites according to the invention do not bind to functional P2X₇receptors, especially receptors wherein Pro210 of P2X₇ is in transconformation.

Typically the antigen binding sites according to the invention bind tonon functional P2X₇ receptors on live cells. In some embodiments, theantigen binding sites do not bind, or bind with very low or undetectableaffinity to non functional receptors on dead or dying cells. Whether anantigen binding site of the invention does or does not bind to a P2X₇receptor can be determined using standard methods known in the art.

In one embodiment, the antigen binding sites according to the inventionbind to P2X₇ receptors on live cells with affinities (K_(D)) in therange of about 1 pM to about 1 uM. Typically, when the antigen bindingsite is part of an IgM the affinity for P2X₇ receptors on live cells isbetween about 1 pM to about 1 nM, preferably about 1 pM to about 50 pM.Typically, when the antigen binding site is part of an IgG the affinityfor P2X₇ receptors on live cells is between about 1 pM to about 1 nM,preferably between about 1 pM to about 100 pM. Typically, when theantigen binding site is part of an Fab the affinity for P2X₇ receptorson live cells is between about 100 pM to about 100 nM, preferably about1 nM to about 100 nM. Typically, when the antigen binding site is partof an scFV the affinity for P2X₇ receptors on live cells is betweenabout 10 nM to about 1 uM, preferably about 10 nM to about 100 nM.Typically, when the antigen binding site is part of an dab the affinityfor P2X₇ receptors on live cells is between about 10 nM to about 10 uM,preferably about 100 nM to about 1 uM.

In certain embodiments, the antigen binding sites of the invention andmolecules comprising same are capable of inducing apoptosis.

In certain embodiments, the antigen binding sites of the invention andmolecules comprising same are capable of inducing caspase activation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Full length human P2X₇ receptor (SEQ ID NO: 1).

FIG. 2. An extracellular domain sequence of P2X₇ receptor. P2X₇ receptor(47-306) (SEQ ID NO: 2) (ECD2) is amino acids 47 to 306 of SEQ ID NO: 1.The amino acids struck-through designate amino acids that are deletedfrom the full length P2X₇ receptor sequence.

FIG. 3. An extracellular domain sequence of P2X₇ receptor. P2X₇ receptor(47-332) (SEQ ID NO:3) (ECD1) is amino acids 47 to 332 of SEQ ID NO: 1.The amino acids struck-through designate amino acids that are deletedfrom the full length P2X₇ receptor sequence.

FIG. 4. Expression vector structure for 2F6 V_(H).

FIG. 5. Expression vector structure for 2F6

FIG. 6. (a) 2F6 scFv sequence with His tag added for detection (SEQ IDNO: 4). The 2F6 scFV sequence shown has the following organisationalstructure (in order from N-terminus to C-terminus)FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a-AAA-Flag®epitope tag (DYKDDDDK)-AAA-His tag. (b) Purification of recombinant 2F6IgG_(2a) by size exclusion HPLC. The recombinant IgG2a was separated byHPLC and an example HPLC chromatogram is shown.

FIG. 7. HP-SEC of 2F6 mIgG2a purification.

FIG. 8. SDS PAGE showing purity of the final antibody product.

FIG. 9. (a) In Vitro Cell Inhibition Assays. The IgM form of theoriginal antibody to the trimer form of the non-functional P2X₇ receptorexpressed on cancer cells was found to inhibit cell growth using theCell Titer Blue Assay. An example is shown in which the control IgMantibody is seen to have no effect on cell growth (left columns) forincreasing concentrations from 2.5 to 40 ug/mL while the 2F6 inhibitedcell growth (right columns) over the same dose range in a 3 day growthassay. (b), (c), (d) Other cell types were similarly inhibited byincubation with the IgM form of the antibody. Growth over 5 days isinhibited to a greater extent than over 3 days. The growth data isplotted relative to the control growth curves obtained using the controlIgM antibody. COLO205 growth was significantly inhibited over 3 days andthe cells were eliminated over 5 days even at low dose of 2.5 ug/mL.This indicates that different cell lines expressing slightly differentlevels of receptor are more or less susceptible to the antibody binding.(e) In contrast, the recombinant IgG2a form of the antibody showedweaker cell inhibition as shown in the following figures obtained over 3days. The cell growth inhibition assay (Cell Titer Blue) showed theIgG_(2a) form of the antibody had reduced tumour cell growth inhibitioncompared with the inhibition elicited using the IgM form of theantibody, in line with the reduced binding affinity of the IgGcontaining as it does, two binding domains rather than ten.

FIG. 10. Blocking Reaction in Cell Killing Assay. The Cell Titer Bluecell growth inhibition assay is used over three day cell growth withMCF-7 breast cancer cell line. Note the viable control cells in theright column have no antibody or peptide. The left hand column is thesignal derived from the cells incubated with 10 ug/mL 2F6 IgM antibodycontaining 500 ug/mL peptide epitope (E200-300 epitope described as200/300 in Figure), sufficient to block the cell growth inhibitionevidenced by the data in the three central columns that show growthinhibition is not affected by the presence of 50 ug/mL, 5 ug/mL or 0ug/mL peptide respectively. Total inhibition of cell growth occurs after5 days of 2F6 exposure.

FIG. 11. Mechanism of Cell Death Induced by 2F6 with Caspase 3/7Activation Associated with Reactivation of Apoptosis. In this experimentthe effect of the Gemcitibine control drug is shown at the left, knownto activate caspases through induction of apoptosis in COLO205 cancercells. In contrast, the absence of drug or antibody has no effect (cellsonly column). The presence of control IgM at doses up to 40 ug/mLsimilarly has no effect on caspase activation while increasing amountsof 2F6 antibody shows a steady increase in Caspase 3/7 activationassociated with apoptosis induction by the antibody over the 3 day timecourse of the experiment.

FIG. 12. Direct Cell Killing by 2F6 IgM. Confocal microscope images ofMCF-7 cells in presence of control IgM antibody (a) and 2F6 IgM (b) for24 h.

FIG. 13. (a) 2-2-1 hFc bound to live 4T1 tumour cells showing somemembranous binding (×40 obj). (b) 2-2-1 hFc bound to dying cells alongwith membranous debris from cells already killed. (c) 2-2-1hFc bound tolive LL tumour cells showing clear membranous binding. (d) 2-2-1hFcbound to membranous debris from cells already killed.

FIG. 14. (a) 2F6 hIgG1 bound to live 4T1 tumour cells showing clearmembranous binding (×40 obj). (b) 2F6 hIgG1 bound only to dying cells.(c) 2F6 hIgG1 bound to live LL tumour cells showing clear membranousbinding. (d) 2F6 hIgG1 bound to dying cells with no binding to adjacentred blood cells expressing function-capable P2X₇ receptor.

FIG. 15. Inhibition of the number of lung metastases by Day 14 in the4T1 syngeneic xenograft model by 2F6hIgG1. The overall reduction intumour volume was 89% with most metastases in the treatment group muchsmaller than in the untreated group.

FIG. 16. Inhibition of the number of lung metastases in the Lewis Lung(LL) syngeneic xenograft model by Day 11. The five groups are theuntreated control (Group 1), sheep polyclonal E200-300 at 10 mg/kg(Group 2), 2F6hIgG1 at 1 mg/kg (Group 3) and at 10 mg/kg (Group 4) andSorafenib at 5 mL/kg daily (Group 5). Both sheep polyclonal and 2F6hIgG1 were equipotent with Sorafenib with 96% inhibition.

FIG. 17. Affinity Maturation of CDR3 Sequences from 2F6. The amino acidsequences of affinity matured scFv/Fab derivatives listed as mutantclones with the wildtype (WT) 2F6 CDR3 sequence at the top of the list.

FIG. 18. ELISA of IgM, IgG2a and Fab Leads. Lead Affinity Matured2F6-Derived Fab ELISA (scale 0.01-12.5 ug/mL for IgM and IgG2a; 0.1-100ug/mL for Fabs). The EC₅₀ values for the original IgM and therecombinant IgG_(2a) were measured to be 0.14 and 1.6 ug/mLrespectively. The WT Fab exhibited a very low EC₅₀ while the leadaffinity matured Fab species selected from ScFv screening (#10, #21, #42and #66) bound much more tightly with an EC₅₀ in the range 2-4 ug/mL orabout 125 times stronger than WT, matching the affinity of the fullyformed IgG_(2a) antibody.

FIG. 19. (a) Flow cytometry results for binding recombinant Fabs to livecolorectal COLO205 tumour cells. A Sigma anti-FLAG secondary antibody(#F4049) was used to detect the binding of the primary antibodies. WT2F6 Fab bound weakly over the same concentration range. The EC50 for thefour lead Fabs is very similar to the values obtained from ELISAmeasurements. (b) Very similar improved binding results were obtainedfor prostate PC3 cells.

FIG. 20. A comparison was made with various preparations of recombinant2F6 IgG_(2a) to determine the relative binding strength of the WTformatted antibody to PC3 cells compared with the affinity matured Fabs.Rockland IgG_(2a) #010-001-332 was used for control to determinebackground (diamond). Binding of the fully formatted WT antibody wascomparable to the binding elicited by the lead Fabs.

FIG. 21. Verification of the lack of binding to functional P2X₇ receptoron human lymphocytes by the lead Fabs was determined by flow cytometry.Sigma anti-FLAG antibody #F4049 was used as the secondary. Abcam HLAantibody was used as a control. No binding was detected above backgroundas determined by the secondary only signal in the left hand column. Theorder or primary antibody from left to right along the x-axis is thesame as the order of the legend from top to bottom.

FIG. 22. Flow cytometry results for binding high affinity purified sheeppolyclonal antibody to PC3 cells showing significantly stronger bindingthan WT 2F6 hIgG2a and indicates the improvements to be expected from arange of affinity matured Fabs once converted to divalent IgG binders.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention. While the invention will be described in conjunction with theembodiments, it will be understood that the intention is not to limitthe invention to those embodiments. On the contrary, the invention isintended to cover all alternatives, modifications, and equivalents,which may be included within the scope of the present invention asdefined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

The invention provides antigen binding sites that are capable of bindingto non-functional P2X₇ receptors expressed by live cells. Thesereceptors are in a higher order oligomeric form. This oligomeric form istwo or more P2X₇ receptor monomers that have associated. Typically, theoligomeric form is a trimer of three P2X₇ receptor monomers. Oneadvantage of the antigen binding sites of the invention which bindhigher order oligomeric P2X₇ forms is that sequestration by monomericforms of the P2X₇ receptor liberated from lysed or apoptotic cells willbe reduced compared to antibodies that only bind monomeric P2X₇receptors.

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth conflicts with any document incorporated hereinby reference, the definition set forth below shall prevail.

“Purinergic receptor” generally refers to a receptor that uses a purine(such as ATP) as a ligand.

“P2X₇ receptor” generally refers to a purinergic receptor formed fromthree protein subunits or monomers, with at least one of the monomershaving an amino acid sequence substantially as shown in SEQ ID NO:1 (seeFIG. 1). “P2X₇ receptor” may be a functional or non functional receptoras described below. “P2X₇ receptor” encompasses naturally occurringvariants of P2X₇ receptor, e.g., wherein the P2X₇ monomers are splicevariants, allelic variants and isoforms including naturally-occurringtruncated or secreted forms of the monomers forming the P2X₇ receptor(e.g., a form consisting of the extracellular domain sequence ortruncated form of it), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants.In certain embodiments of the invention, the native sequence P2X₇monomeric polypeptides disclosed herein are mature or full-length nativesequence polypeptides comprising the full-length amino acids sequenceshown in SEQ ID NO:1. In certain embodiments the P2X₇ receptor may havean amino acid sequence that is modified, for example various of theamino acids in the sequence shown in SEQ ID NO:1 may be substituted,deleted, or a residue may be inserted.

“Functional P2X₇ receptor” generally refers to a form of the P2X₇receptor having a binding site or cleft for binding to ATP. When boundto ATP, the receptor forms a pore-like structure that enables theingress of calcium ions into the cytosol, one consequence of which maybe programmed cell death. In normal homeostasis, expression offunctional P2X₇ receptors is generally limited to cells that undergoprogrammed cell death such as thymocytes, dendritic cells, lymphocytes,macrophages and monocytes. There may also be some expression offunctional P2X₇ receptors on erythrocytes.

“Non functional P2X₇ receptor” generally refers to a form of a P2X₇receptor in which one or more of the monomers has a cis isomerisation atPro210 (according to SEQ ID NO:1). The isomerisation may arise from anymolecular event that leads to misfolding of the monomer, including forexample, mutation of monomer primary sequence or abnormal posttranslational processing. One consequence of the isomerisation is thatthe receptor is unable to bind to ATP. In the circumstances, thereceptor cannot form a pore and this limits the extent to which calciumions may enter the cytosol. Non functional P2X₇ receptors are expressedon a wide range of epithelial and haematopoietic cancers.

“Extracellular domain” (ECD) used herein are P2X₇ receptor (47-306) (SEQID NO: 2, see FIG. 2) (ECD2) and P2X₇ receptor (47-332) (SEQ ID NO:3)(ECD1). P2X₇ receptor (47-306) (SEQ ID NO: 2) is amino acids 47 to 306of SEQ ID NO: 1. P2X₇ receptor (47-332) (SEQ ID NO:3, see FIG. 3) isamino acids 47 to 332 of SEQ ID NO: 1.

“Antibodies” or “immunoglobulins” or “Igs” are gamma globulin proteinsthat are found in blood, or other bodily fluids of verterbrates thatfunction in the immune system to bind antigen, hence identifying andneutralizing foreign objects.

Antibodies are generally a heterotetrameric glycoprotein composed of twoidentical light (L) chains and two identical heavy (H) chains. Each Lchain is linked to a H chain by one covalent disulfide bond. The two Hchains are linked to each other by one or more disulfide bonds dependingon the H chain isotype. Each H and L chain also has regularly spacedintrachain disulfide bridges.

H and L chains define specific Ig domains. More particularly, each Hchain has at the N-terminus, a variable domain (V_(H)) followed by threeconstant domains (C_(H)) for each of the α and γ chains and four C_(H)domains for μ and ε isotypes. Each L chain has at the N-terminus, avariable domain (V_(L)) followed by a constant domain (C_(L)) at itsother end. The V_(L) is aligned with the V_(H) and the C_(L) is alignedwith the first constant domain of the heavy chain (C_(H)1).

Antibodies can be assigned to different classes or isotypes. There arefive classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ, and μ respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The Lchain from any vertebrate species can be assigned to one of two clearlydistinct types, called kappa and lambda, based on the amino acidsequences of their constant domains.

The constant domain includes the Fc portion which comprises thecarboxy-terminal portions of both H chains held together by disulfides.The effector functions of antibodies such as ADCC are determined bysequences in the Fc region, which region is also the part recognized byFc receptors (FcR) found on certain types of cells.

The pairing of a V_(H) and V_(L) together forms a “variable region” or“variable domain” including the amino-terminal domains of the heavy orlight chain of the antibody. The variable domain of the heavy chain maybe referred to as “VH.” The variable domain of the light chain may bereferred to as “VL.” The V domain contains an antigen binding site whichaffects antigen binding and defines specificity of a particular antibodyfor its particular antigen. V regions span about 110 amino acid residuesand consist of relatively invariant stretches called framework regions(FRs) (generally about 4) of 15-30 amino acids separated by shorterregions of extreme variability called “hypervariable regions” (generallyabout 3) that are each 9-12 amino acids long. The FRs largely adopt a0-sheet configuration and the hypervariable regions form loopsconnecting, and in some cases forming part of, the 0-sheet structure.

“Hypervariable region”, “HVR”, or “HV” refers to the regions of anantibody variable domain which are hypervariable in sequence and/or formstructurally defined loops. Generally, antibodies comprise sixhypervariable regions; three in the VH(HI, H2, H3), and three in the VL(LI, L2, L3). A number of hypervariable region delineations are in useand are encompassed herein. The Kabat Complementarity DeterminingRegions (CDRs) are based on sequence variability and are the mostcommonly used (Kabat et al, Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)).

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues herein defined.

“A peptide for forming an antigen binding site” generally refers to apeptide that may form a conformation that confers the specificity of anantigen for antigen. Examples include whole antibody or whole antibodyrelated structures, whole antibody fragments including a variabledomain, variable domains and fragments thereof, including light andheavy chains, or fragments of light and heavy chains that include somebut not all of hypervariable regions or constant regions.

An “intact” or “whole” antibody is one which comprises anantigen-binding site as well as a C_(L) and at least heavy chainconstant domains, C_(H)I, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variant thereof.

“Whole antibody related structures” include multimerized forms of wholeantibody.

“Whole antibody fragments including a variable domain” include Fab,Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies,single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

The Fab fragment consists of an entire L chain along with the variableregion domain of the H chain (V_(H)), and the first constant domain ofone heavy chain (C_(H)I). Each Fab fragment is monovalent with respectto antigen binding, it has a single antigen-binding site.

A Fab′ fragment differs from Fab fragments by having additional fewresidues at the carboxy terminus of the C_(H)I domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group.

A F(ab′)₂ fragment roughly corresponds to two disulfide linked Fabfragments having divalent antigen-binding activity and is still capableof cross-linking antigen.

An “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association.

In a single-chain Fv (scFv) species, one heavy- and one light-chainvariable domain can be covalently linked by a flexible peptide linkersuch that the light and heavy chains can associate in a “dimeric”structure analogous to that in a two-chain Fv species. From the foldingof these two domains emanate six hypervariable loops (3 loops each fromthe H and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody.

“Single-chain FV” also abbreviated as “sFv” or “scFV” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedto form a single polypeptide chain. Preferably, the scFv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains which enables the scFv to form the desired structure for antigenbinding.

A “single variable domain” is half of an Fv (comprising only three CDRsspecific for an antigen) that has the ability to recognize and bindantigen, although at a lower affinity, than the entire binding site

“Diabodies” refers to antibody fragments with two antigen-binding sites,which fragments comprise a heavy-chain variable domain (VH) connected toa light-chain variable domain (VL) in the same polypeptide chain(VH-VL). The small antibody fragments are prepared by constructing sFvfragments (see preceding paragraph) with short linkers (about 5-10residues) between the V_(H) and V_(L) domains such that interchain butnot intra-chain pairing of the V domains is achieved, resulting in abivalent fragment, i.e., fragment having two antigen-binding sites.

Diabodies may be bivalent or bispecific. Bispecific diabodies areheterodimers of two “crossover” sFv fragments in which the V_(H) andV_(L) domains of the two antibodies are present on different polypeptidechains. Triabodies and tetrabodies are also generally know in the art.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its pre-existing environment.Contaminant components are materials that would interfere withtherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes.

A “human antibody” refers to an antibody which possesses an amino acidsequence which corresponds to that of an antibody produced by a humanand/or has been made using any of the techniques for making humanantibodies as disclosed herein. This definition of a human antibodyspecifically excludes a humanized antibody comprising non-humanantigen-binding residues. Human antibodies can be produced using varioustechniques known in the art, including phage-display libraries. Humanantibodies can be prepared by administering the antigen to a transgenicanimal that has been modified to produce such antibodies in response toantigenic challenge, but whose endogenous loci have been disabled.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit, dog, cat or non-human primate having the desiredantibody specificity, affinity, and capability. In some instances,framework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

“Monoclonal antibody” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic site or determinant on the antigen. In addition to theirspecificity, the monoclonal antibodies are advantageous in that they maybe synthesized uncontaminated by other antibodies. Monoclonal antibodiesmay be prepared by the hybridoma methodology, or may be made usingrecombinant DNA methods in bacterial, eukaryotic animal or plant cells.The “monoclonal antibodies” may also be isolated from phage antibodylibraries.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity. Chimeric antibodies of interest herein include“primatized” antibodies comprising variable domain antigen-bindingsequences derived from a non-human primate (e.g. Old World Monkey, Apeetc), and human constant region sequences.

The term “anti-P2X₇ receptor antibody” or “an antibody that binds toP2X₇ receptor” refers to an antibody that is capable of binding P2X₇receptor with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting P2X₇ receptor,typically non functional P2X₇ receptor. Preferably, the extent ofbinding of an P2X₇ receptor antibody to an unrelated receptor protein isless than about 10% of the binding of the antibody to P2X₇ receptor asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that binds to P2X₇ receptor has a dissociation constant (Kd) of<1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. An anti non functional P2X₇receptor antibody is generally one having some or all of theseserological characteristics and that binds to non functional P2X₇receptors but not to functional P2X₇ receptors.

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen).Generally, “binding affinity” refers to intrinsic binding affinity whichreflects a 1:1 interaction between members of a binding pair (e.g.,antibody and antigen). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (Kd). Affinitycan be measured by common methods known in the art, including thosedescribed herein. Low-affinity antibodies generally bind antigen slowlyand tend to dissociate readily, whereas high-affinity antibodiesgenerally bind antigen faster and tend to remain bound longer. A varietyof methods of measuring binding affinity are known in the art, any ofwhich can be used for purposes of the present invention.

As used herein, the properties of amino acids are defined in thefollowing table:

Amino Acid 3-letter code 1-letter code Properties Alanine Ala Aaliphatic hydrophobic neutral Arginine Arg R polar hydrophilic charged(+) Asparagine Asn N polar hydrophilic neutral Aspartate Asp D polarhydrophilic charged (−) Cysteine Cys C polar hydrophobic neutralGlutamine Gln Q polar hydrophilic neutral Glutamate Glu E polarhydrophilic charged (−) Glycine Gly G aliphatic neutral Histidine His Haromatic polar hydrophilic charged (+) Isoleucine Ile I aliphatichydrophobic neutral Leucine Leu L aliphatic hydrophobic neutral LysineLys K polar hydrophilic charged (+) Methionine Met M hydrophobic neutralPhenylalanine Phe F aromatic hydrophobic neutral Proline Pro Phydrophobic neutral Serine Ser S polar hydrophilic neutral Threonine ThrT polar hydrophilic neutral Tryptophan Trp W aromatic hydrophobicneutral Tyrosine Tyr Y aromatic polar hydrophobic Valine Val V aliphatichydrophobic neutral

The inventors have determined the CDR sequences of a number of variabledomain clones that they have found to bind to non-functional P2X₇receptor. These CDR sequences, are shown in Table 1a below.

In one embodiment there is provided a peptide having a sequence as shownin Table 1a or b. These peptides are particularly useful forconstructing antigen binding sites, variable domains, antibodies andrelated fragments.

TABLE 1a CDR sequences CDR1 CDR2 CDR3 KASQNVGTNVA SASFRYS(charged/polar/aromatic) (charged/aromatic) (SEQ ID NO: 5) (SEQ IDXXXY (aromatic/aliphatic)(charged/neutral) NO: 6) (neutral/aliphatic)N(Y/F))XXXY(Y(Y/F)EX N(Y/F)(neutral)(charged)(neutral)Y(Y/F)E(neutral)NFLESYFEA (SEQ ID NO: 7)N(Y/F)(charged)(neutral)(charged)Y(Y/F)E(neutral) NYRGDYYET(SEQ ID NO: 8) H(aromatic)XXXYYNI H(Y/F)(neutral)(charged)(neutral)YYNIH(Y/F)(neutral)(charged)(charged)YYNI HYSKEYYNI (SEQ ID NO: 9) HFQRGYYNI(SEQ ID NO: 10) (Y/N)(aromatic)XXXYY(charged)(neutral)(Y/N)(aromatic)(neutral)(neutral)(neutral)YYDV(Y/N)(aromatic)(neutral)(neutral)(neutral)YYEV YFPLVYYDV (SEQ ID NO: 11)NYLPMYYEV (SEQ ID NO: 12) Y(charged)XXXY(neutral)(neutral)(neutral)NFKLMYYNV (SEQ ID NO: 13)(charged/polar/aromatic)(aromatic)(charged/neutral)(charged)(charged/neutral)Y(aromatic)(charged) (neutral)(charged/polar/aromatic)(F/Y)(charged/neutral)(R/K)(charged/neutral)(Y)(Y/F)(E/D)V (H/N)(F/Y)(S/D)(R/K)(G/K)Y(Y/F)DVHFSRGYYDV (SEQ ID NO: 14) HYIKVYYEA (SEQ ID NO: 15) HYSSRFFEV(SEQ ID NO: 16) NFRVMFFKA (SEQ ID NO: 17) HYSSRFFEV (SEQ ID NO: 18)YHVIQYLGP (SEQ ID NO: 19) DFTVPFYNA (SEQ ID NO: 20) NYDKKYFDV(SEQ ID NO: 21) YFPLVYYDV (SEQ ID NO: 22) HYSSRFFDV (SEQ ID NO: 34)SYYMS AINSNGGS (charged/polar/aromatic) (charged/aromatic)(SEQ ID NO: 23) YYPTVKG XXXY (aromatic/aliphatic)(charged/neutral)(SEQ ID (neutral/aliphatic) NO: 24) N(Y/F)XXXY(Y/F)EXN(Y/F)(neutral)(charged)(neutral)Y(Y/F)E(neutral) NFLESYFEAN(Y/F)(charged)(neutral)(charged)Y(Y/F)E(neutral) NYRGDYYETH(aromatic)XXXYYNI H(Y/F)(neutral)(charged)(neutral)YYNIH(Y/F)(neutral)(charged)(charged)YYNI HYSKEYYNI HFQRGYYNI(Y/N)(aromatic)XXXYY(charged)(neutral)(Y/N)(aromatic)(neutral)(neutral)(neutral)YYDV(Y/N)(aromatic)(neutral)(neutral)(neutral)YYEV YFPLVYYDV NYLPMYYEVY(charged)XXXY(neutral)(neutral)(neutral) YHVIQYLGP NFKLMYYNV(charged/polar/aromatic)(aromatic)(charged/neutral)(charged)(charged/neutral)Y(aromatic)(charged) (neutral)(charged/polar/aromatic)(F/Y)(charged/neutral)(R/K)(charged/neutral)(Y)(Y/F)(E/D)V (H/N)(F/Y)(S/D)(R/K)(G/K)Y(Y/F)DVHFSRGYYDV HYIKVYYEA HYSSRFFEV NFRVMFFKA HYSSRFFEV DFTVPFYNA NYDKKYFDVYFPLVYYDV HYSSRFFDV

TABLE 1b Antigen binding site V_(H) V_(L) scFV 2F6 (WT) DVKLVESGGGLVKLGMADIVMTQSQKFMSTSV MADIVMTQSQKFM GSLKLSCAASGFTFSS GDRVSVTCKASQNVGTNSTSVGDRVSVTCK YYMSWVRQTPEKRLE VAWYQQKPGQSPKALIY ASQNVGTNVAWYLVAAINSNGGSTYYPD SASFRYSGVPDRFTGSG QQKPGQSPKALIY TVKGRFTISRDNAKNTSGTDFTLTISNVQSEDLA SASFRYSGVPDRF LYLQMSSLKSEDTAFY EFFCQQYNSYPFTFGSGTGSGSGTDFTLTIS YCTRHYSSRFFDVWG TRLEIK NVQSEDLAEFFCQ AGTTVTVSS(SEQ ID NO: 36) QYNSYPFTFGSGT (SEQ ID NO: 35) RLEIKGGGGSGGG GSGGGGSDVKLVESGGGLVKLGGSL KLSCAASGFTFSS YYMSWVRQTPEK RLELVAAINSNGG STYYPDTVKGRFTISRDNAKNTLYLQM SSLKSEDTAFYYC TRHYSSRFFDVW GAGTTVTVSS (SEQ ID NO: 37)Mutant DVKLVESGGGLVKLG MADIVMTQSQKFMSTSV MADIVMTQSQKFM #18GSLKLSCAASGFTFSS GDRVSVTCKASQNVGTN STSVGDRVSVTCK YYMSWVRQTPEKRLEVAWYQQKPGQSPKALIY ASQNVGTNVAWY LVAAINSNGGSTYYPD SASFRYSGVPDRFTGSGQQKPGQSPKALIY TVKGRFTISRDNAKNT SGTDFTLTISNVQSEDLA SASFRYSGVPDRFLYLQMSSLKSEDTAFY EFFCQQYNSYPFTFGSG TGSGSGTDFTLTIS YCTRHFSRGYYDVWG TRLEIKNVQSEDLAEFFCQ AGTTVTVSS QYNSYPFTFGSGT (SEQ ID NO: 38) RLEIKGGGGSGGGGSGGGGSDVKLV ESGGGLVKLGGSL KLSCAASGFTFSS YYMSWVRQTPEK RLELVAAINSNGGSTYYPDTVKGRFTI SRDNAKNTLYLQM SSLKSEDTAFYYC TRHFSRGYYDVW GAGTTVTVSS(SEQ ID NO: 39) Mutant DVKLVESGGGLVKLG MADIVMTQSQKFMSTSV MADIVMTQSQKFM#78 GSLKLSCAASGFTFSS GDRVSVTCKASQNVGTN STSVGDRVSVTCK YYMSWVRQTPEKRLEVAWYQQKPGQSPKALIY ASQNVGTNVAWY LVAAINSNGGSTYYPD SASFRYSGVPDRFTGSGQQKPGQSPKALIY TVKGRFTISRDNAKNT SGTDFTLTISNVQSEDLA SASFRYSGVPDRFLYLQMSSLKSEDTAFY EFFCQQYNSYPFTFGSG TGSGSGTDFTLTIS YCTRNYDKKYFDVWG TRLEIKNVQSEDLAEFFCQ AGTTVTVSS QYNSYPFTFGSGT (SEQ ID NO: 40) RLEIKGGGGSGGGGSGGGGSDVKLV ESGGGLVKLGGSL KLSCAASGFTFSS YYMSWVRQTPEK RLELVAAINSNGGSTYYPDTVKGRFTI SRDNAKNTLYLQM SSLKSEDTAFYYC TRNYDKKYFDVW GAGTTVTVSS(SEQ ID NO: 41)

In certain embodiments the antigen binding site is one having at least75%, preferably 80%, more preferably 85%, more preferably 90%, morepreferably 95%, more preferably 96%, 97%, 98% or 99% identity to anantigen binding site described above.

In certain embodiments the CDR is one having at least 75%, preferably80%, more preferably 85%, more preferably 90%, more preferably 95%, morepreferably 96%, 97%, 98% or 99% identity to a CDR shown in Table 1a.

In certain embodiments the antigen binding site comprises or consists ofa V_(H), V_(L) or scFv sequence shown in Table 1b or has a sequence thathas 75%, preferably 80%, more preferably 85%, more preferably 90%, morepreferably 95%, more preferably 96%, 97%, 98% or 99% identity to aV_(H), V_(L) or scFV sequence described in Table 1b.

In other embodiments there is provided an antigen binding site or CDRand/or FR having a sequence as described above and including one or moremutations for increasing the affinity of said site for binding to ananti-P2X₇ receptor. The mutation may result in a substitution, insertionor deletion of a residue in one or more of CDR1, CDR2 or CDR3, or one ormore or FR1, FR2, FR3 or FR4.

In certain embodiments antigen binding sites of the invention andmolecules comprising same bind to an epitope that is exclusivelyexpressed on non ATP-binding P2X₇ receptors (otherwise known as“non-functional receptors”). The epitope and peptides forming it havebeen found to be useful for generating monoclonal antibodies that bindto non-functional P2X₇ receptors expressed on live cells.

Live cell binding is important because the expression of the nonfunctional P2X₇ receptor in or on cells, examples being epithelialcells, is believed to be a biomarker of many cancers such as epithelialcancers and other conditions. Accordingly, with monoclonal antibodiesthat bind live cells it becomes possible to provide systemictherapeutics either in the form of the antibody itself, or anantibody—cytotoxic agent conjugate—to a wide range of diseasescharacterised by expression of non functional P2X₇ receptors. It alsobecomes possible to provide for in vivo imaging and diagnosis ormonitoring of diseases characterised by expression of non functionalP2X₇ receptors.

The epitope is found only on the P2X₇ receptor i.e. the trimer formedfrom P2X₇ monomers. More particularly, the epitope spans adjacent P2X₇monomers in the trimeric P2X₇ receptor. Individual P2X₇ monomers thatare not aligned as in a non functional trimeric receptor therefore donot contain the epitope. This advantageously permits one to stagetumours. This is more difficult to do with antibodies that bind to bothmonomeric P2X₇ and the trimeric receptor.

Thus in certain embodiments the antigen binding sites of the inventionbind to an epitope of a P2X₇ receptor

the epitope being formed of:

-   -   a first region in the form of a region of a first monomer of a        P2X₇ receptor; and    -   a second region in the form of a region of a second monomer of        the receptor;        wherein the first and second regions are formed in the receptor        by cis isomerisation of a residue at position 210 of SEQ ID No:        1 of a monomer of the receptor;        and wherein the first and second regions are arranged adjacent        each other in the receptor thereby permitting binding of an        antigen binding site of an anti-P2X₇ antibody to the first and        second regions forming the epitope.

Typically the epitope is a conformational epitope. In these embodiments,the first and second regions each define a molecular space that eachinclude one or more residues of SEQ ID No: 1. Typically the first regionis one that defines a molecular space including one or more of theresidues of SEQ ID No: 1: that are exposed for binding to an antigenbinding site of an antibody as a consequence of cis isomerisation ofPro210 of a monomer having a sequence shown in SEQ ID No: 1. Theseresidues include Gly 200, His 201, Asn 202, Tyr 203, Thr 204, Thr 205,Arg 206, Asn 207, Ile 208, Leu 209 and Pro210. In one embodiment thefirst region includes at least one of these residues. Typically thefirst region includes at least 4 of these residues, although it may beless, for example, 2 or 3, depending on how many residues are presentedin the second region. In one embodiment, the first region includes atleast 1 pair of residues shown in the Table 2 below:

TABLE 2 His 201 Asn 202 Tyr 203 Thr 204 Thr 205 Arg 206 Asn 207 Ile 208Leu 209 Gly 200 Gly 200 Gly 200 Gly 200 Gly 200 Gly 200 Gly 200 Gly 200Gly 200 Asn 202 Tyr 203 Thr 204 Thr 205 Arg 206 Asn 207 Ile 208 Leu 209His 201 His 201 His 201 His 201 His 201 His 201 His 201 His 201 Tyr 203Thr 204 Thr 205 Arg 206 Asn 207 Ile 208 Leu 209 Asn 202 Asn 202 Asn 202Asn 202 Asn 202 Asn Asn 202 202 Thr 204 Thr 205 Arg 206 Asn 207 Ile 208Leu 209 Tyr 203 Tyr 203 Tyr 203 Tyr 203 Tyr 203 Tyr 203 Thr 205 Arg 206Asn 207 Ile 208 Leu 209 Thr 204 Thr 204 Thr 204 Thr 204 Thr 204 Arg 206Asn 207 Ile 208 Leu 209 Thr 205 Thr 205 Thr 205 Thr 205 Asn 207 Ile 208Leu 209 Arg 206 Arg Arg 206 206 Ile 208 Leu 209 Asn Asn 207 207 Leu 209Ile 208

In certain embodiments the first region includes 2 or more pairs ofresidues shown in Table 2.

The first region may additionally contain one or more peripheralresidues that are intimately involved in formation of the ATP bindingsite on the larger of the two extracellular domain folds. These are Lys193, Phe 275 and Arg 294. Arg 125 is located in the smaller of the twoextracellular domain folds. Thus in certain embodiments, the firstregion further includes one or more of the following residues of SEQ IDNo: 1: Arg 125, Lys 193, Phe 275 and Arg 294. It will be understood thatthe first region does not consist of these residues alone. That is thefirst region, as discussed above, defines a molecular space includingone or more of the residues of SEQ ID No: 1: that are exposed forbinding to an antigen binding site of an antibody as a consequence ofcis isomerisation of Pro210 of a monomer having a sequence shown in SEQID No: 1. In this context, the Arg 125, Lys 193, Phe 275 and Arg 294 areonly provided in addition, but not alternate to for example one or moreof the residues Gly 200, His 201, Asn 202, Tyr 203, Thr 204, Thr 205,Arg 206, Asn 207, Ile 208, Leu 209.

Typically the second region is one that defines a molecular spaceincluding one or more of the residues of SEQ ID No: 1: that are exposedfor binding to an antigen binding site of an antibody as a consequenceof cis isomerisation of Pro210 of a monomer having a sequence shown inSEQ ID No: 1. These residues include Lys 297, Tyr 298, Tyr 299, Lys 300,Glu 301, Asn 302, Asn 303, Val 304, Glu 305 and Lys 306. In oneembodiment the second region includes at least one of these residues.Typically the second region includes at least 4 of these residues,although it may be less, for example, 2 or 3, depending on how manyresidues are presented in the first region. In one embodiment, thesecond region includes at least 1 pair of residues shown in the Table 3below:

TABLE 3 Tyr 298 Tyr 299 Lys 300 Glu 301 Asn 302 Asn 303 Val 304 Glu 305Lys 306 Lys 297 Lys 297 Lys 297 Lys 297 Lys 297 Lys 297 Lys 297 Lys 297Lys 297 Tyr 298 Tyr 298 Tyr 298 Tyr 298 Tyr 298 Tyr 298 Tyr 298 Tyr 298Tyr 299 Lys 300 Glu 301 Asn 302 Asn 303 Val 304 Glu 305 Lys 306 Tyr 299Tyr 299 Tyr 299 Tyr 299 Tyr 299 Tyr 299 Tyr 299 Glu 301 Glu 301 Asn 302Asn 303 Val 304 Glu 305 Lys 306 Lys 300 Lys 300 Lys 300 Lys 300 Lys 300Lys 300 Glu 301 Asn 302 Asn 303 Val 304 Glu 305 Lys 306 Glu 301 Glu 301Glu 301 Glu 301 Glu 301 Asn 302 Asn 303 Val 304 Glu 305 Lys 306 Asn 302Asn 302 Asn 302 Asn 302 Asn 303 Val 304 Glu 305 Lys 306 Asn 303 Asn 303Asn 303 Val 304 Glu 305 Lys 306 Val 304 Val 304 Glu 305 Lys 306 Glu 305Lys 306

In certain embodiments the second region includes 2 or more pairs ofresidues shown in Table 3.

The second region may additionally contain one or more peripheralresidues that are intimately involved in formation of the ATP bindingsite. These are Arg 307 and Lys 311. Thus in certain embodiments, thesecond region further includes Arg 307 and/or Lys 311. It will beunderstood that the second region does not consist of these residuesalone. That is, the second region, as discussed above, defines amolecular space including one or more of the residues of SEQ ID No: 1:that are exposed for binding to an antigen binding site of an antibodyas a consequence of cis isomerisation of Pro210 of a monomer having asequence shown in SEQ ID No: 1. In this context, the Arg 307 and Lys 311are only provided in addition, but not alternate to for example one ormore of the residues Lys 297, Tyr 298, Tyr 299, Lys 300, Glu 301, Asn302, Asn 303, Val 304, Glu 305 and Lys 306.

In certain embodiments, the epitope is, or includes a linear epitope.Examples include where the first region includes one of the followingsequences of SEQ ID No: 1 in Table 4:

TABLE 4 Gly 200 to Tyr 203 His 201 to Thr 204 Asn 202 to Thr 205 Tyr 203to Arg 206 Thr 204 to Asn 207 Thr 205 to Ile 208 Arg 206 to Leu 209

In these embodiments, the second region of the epitope may include oneof the following sequences of SEQ ID No: 1 in Table 5:

TABLE 5 Lys 297 to Lys 300 Tyr 298 to Glu 301 Tyr 299 to Asn 301 Lys 300to Asn 303 Glu 301 to Val 304 Asn 301 to Glu 305 Asn 303 to Lys 306

In certain embodiments, the first region contains more residues than thesecond region. In other embodiments, the second region contains moreresidues than the first region.

The first region and second region may each contain from about 4 toabout 10 residues, for example 5, 6, 7, 8 or 9 residues. Where there aremore residues in the second region, there may be fewer residues in thefirst region, ie less than 4, for example 2 or 3. The same applies viceversa.

As described herein, the first and second regions are arranged adjacenteach other in the receptor thereby permitting binding of an antigenbinding site of an anti-P2X₇ antibody to the first and second regionsforming the epitope. In more detail, the inventors have found thatalthough located on separate monomers, the first and second regions incombination form an epitope that can be bound by a single antigenbinding site of an antibody. Generally, the first and second regions ofthe epitope are spaced apart no more than about 40 Angstroms. If thedistance is greater than this, the antibody binding affinity tends todecrease as the antigen binding site is required to traverse a largerdistance across the monomers within the receptor in which case fewerresidues are bound. Generally the first and second regions are spacedapart about 10 Angstroms, although greater distances less than 40Angstroms are possible such as 15, 20, 25, 30, 35 Angstroms.

The epitope described herein may be provided in a substantially purifiedor isolated form, for example as a fragment of a naturally occurringP2X₇ receptor or as a synthetic or recombinant P2X₇ receptor.

Marks et al. (1992) BioTechnology 10:779, which describes affinitymaturation by VH and VL domain shuffling; Barbas et al. (1994) Proc Nat.Acad. Sci. USA 9 1:3809; Schier et al. (1995) Gene 169:147-155; Yeltonet al. (1995) J. Immunol. 155:1994; Jackson et al (1995), J. Immunol.154(7):3310; and Hawkins et al, (1992) J. Mol. Biol. 226:889, whichdescribe random mutagenesis of hypervariable region and/or frameworkresidues, are examples of procedures known in the art for affinitymaturation of antigen binding sites. In certain embodiments, a nucleicacid encoding one or more of the sequences shown in Table 1a or b ismutagenized to create a diverse library of sequences. The library isthen screened against a target including an epitope of a non functionalP2X₇ receptor. An exemplary method is shown in the Examples herein.

In another embodiment there is provided an antigen binding site asdescribed above wherein an amino acid sequence forming one or more ofFR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 is derived from a human sequenceor in the form of a human sequence.

The antigen binding site may be presented in a humanized form includingnon-human (e.g., murine) and human immunoglobulin sequences. Typicallyall but the CDR sequences of the antigen binding site are from anon-human species such as mouse, rat or rabbit. In some instances,framework residues of the antigen binding site may also be non human.Where the antigen binding site is provided in the form of a wholeantibody, typically at least a portion of an immunoglobulin constantregion (Fc) is human, thereby allowing various human effector functions.

Methods for humanizing non-human antigen binding sites are well known inthe art, examples of suitable processes including those in Jones et al.,(1986) Nature, 321:522; Riechmann et al., (1988) Nature, 332:323;Verhoeyen et al., (1988) Science, 239:1534.

Phage display methods described herein using antibody libraries derivedfrom human immunoglobulin sequences are useful for generating humanantigen binding sites and human antibodies.

Also, transgenic mammals that are incapable of expressing functionalendogenous immunoglobulins, but which can express human immunoglobulingenes can be used. These mice may be generated by random or targetedinsertion of the human heavy and light chain immunoglobulin genes intoembryonic stem cells. The host heavy and light chain immunoglobulingenes may be rendered non-functional by the insertion or by some otherrecombination event, for example by homozygous deletion of the host JHregion. The transfected embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice that are thenbred to produce homozygous offspring that express human antigen bindingsites. After immunization with a P2X₇ epitope, human monoclonalantibodies can be obtained. One benefit of transgenic animal systems isthat it is possible to produce therapeutically useful isotypes becausethe human immunoglobulin transgenes rearrange during B-celldifferentiation and subsequently undergo class switching and somaticmutation in the transgenic mice.

Variable domains including CDRs and FRs of the invention may have beenmade less immunogenic by replacing surface-exposed residues so as tomake the antibody appear as self to the immune system. Padlan, E. A.,1991, Mol. Immunol. 28, 489 provides an exemplary method. Generally,affinity is preserved because the internal packing of amino acidresidues in the vicinity of the antigen binding site remains unchangedand generally CDR residues or adjacent residues which influence bindingcharacteristics are not to be substituted in these processes.

In another embodiment there is provided an anti P2X₇ receptorimmunoglobulin variable domain, antibody, Fab, dab or scFv including anantigen binding site as described above.

Lower molecular weight antibody fragments, as compared with wholeantibodies may have improved access to solid tumors and more rapidclearance which may be particularly useful in therapeutic and in vivodiagnostic applications.

Various techniques have been developed for the production of antibodyfragments including proteolytic digestion of intact antibodies andrecombinant expression in host cells. With regard to the latter, asdescribed below, Fab, Fv and scFv antibody fragments can all beexpressed in and secreted from E. coli, antibody fragments can beisolated from the antibody phage libraries and Fab′-SH fragments can bedirectly recovered from E. coli and chemically coupled to form F(ab′)2fragments. In another approach, F(ab′)2 fragments are isolated directlyfrom recombinant host cell culture.

In certain embodiments, the antigen binding site is provided in the formof a single chain Fv fragment (scFv). Fv and scFv are suitable forreduced nonspecific binding during in vivo use as they have intactcombining sites that are devoid of constant regions. Fusion proteinsincluding scFv may be constructed to yield fusion of an effector proteinat either the amino or the carboxy terminus of an scFv. Preferably thescFV is in the form of a V_(H) domain fused by a linker to a V_(L)domain. In one embodiment the linker is at least 15 amino acids inlength. Typically, the linker is at least 10 amino acids in length. Inone embodiment the linker is comprised of generally glycine or serineresidues. Typically, the linker is GGGGSGGGGSGGGGS.

In one embodiment the scFV has the sequence:

MADIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASFRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEFFCQQYNSYPFTFGSGTRLEIKGGGGSGGGGSGGGGSDVKLVESGGGLVKLGGSLKLSCAASGFTFSSYYMSWVRQTPEKRLELVAAINSNGGSTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAFYYCTRHYSSRFFDVWGAGTTVTVSS

In another embodiment there is provided a diabody or triabody or othermultispecific antibody including an antigen binding site as describedabove. Multispecific antibodies may be assembled using polypeptidedomains that allow for multimerization. Examples include the CH2 and CH3regions of the Fc and the CH1 and Ckappa/lambda regions. Other naturallyoccurring protein multimerization domains may be used including leucinezipper domain (bZIP), helix-loop-helix motif, Src homology domain (SH2,SH3), an EF hand, a phosphotyrosine binding (PTB) domain, or otherdomains known in the art.

In another embodiment there is provided a fusion domain or heterologousprotein including an antigen binding site, immunoglobulin variabledomain, antibody, Fab, dab, scFv, diabody or triabody as describedabove.

A heterologous polypeptide may be recombinantly fused or chemicallyconjugated to an N- or C-terminus of an antigen binding site or moleculecontaining same of the invention.

The heterologous polypeptide to which the antibody or antigen bindingsite is fused may be useful to target to the P2X₇ receptor expressingcells, or useful to some other function such as purification, orincreasing the in vivo half life of the polypeptides, or for use inimmunoassays using methods known in the art.

In preferred embodiments, a marker amino acid sequence such as ahexa-histidine peptide is useful for convenient purification of thefusion protein. Others include, but are not limited to, the “HA” tag,which corresponds to an epitope derived from the influenza hemagglutininprotein and the “flag” tag. For example, an scFv of the invention may beboth flag tagged and His tagged with following sequence:

MADIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASFRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEFFCQQYNSYPFTFGSGTRLEIKGGGGSGGGGSGGGGSDVKLVESGGGLVKLGGSLKLSCAASGFTFSSYYMSWVRQTPEKRLELVAAINSNGGSTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAFYYCTRHYSSRFFDVWGAGTTVTVSSAAADYKDD DDKAAAHHHHHH

Further, the antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody or triabody of the invention may bemodified by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc.

Antigen binding sites of the invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. Antigen binding sites of the invention may bemodified by natural processes, such as posttranslational processing, orby chemical modification techniques which are well known in the art.Such modifications are well described in basic texts, as well as inresearch literature. Modifications can occur anywhere in the antigenbinding site, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini, or on moieties such as carbohydrates.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given antigenbinding site. Also, a given antigen binding site may contain many typesof modifications. An antigen binding site may be branched, for example,as a result of ubiquitination, and they may be cyclic, with or withoutbranching. Cyclic; branched, and branched cyclic antigen binding sitesmay result from posttranslation natural processes or may be made bysynthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.

In another embodiment there is provided a conjugate in the form of anantigen binding site, immunoglobulin variable domain, antibody, Fab,dab, scFsv, diabody, triabody or fusion protein as described aboveconjugated to a cytotoxic agent such as a chemo therapeutic agent, adrug, a growth inhibitory agent, a toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a label such as a radioactive isotope (i.e., a radioconjugate). In another aspect, the invention further provides methods ofusing the immunoconjugates. In one aspect, an immunoconjugate comprisesany of the above variable domains covalently attached to a cytotoxicagent or a detectable agent.

In another embodiment there is provided an antibody for binding to anantigen binding site of an immunoglobulin variable domain, antibody,Fab, dab, scFv, diabody, triabody, fusion protein or conjugate asdescribed above.

In another embodiment there is provided a nucleic acid encoding anantigen binding site, immunoglobulin variable domain, antibody, Fab,dab, scFv, diabody, triabody, fusion protein or conjugate as describedabove.

A polynucleotide encoding an CDR or FR according to any one of thegeneral formulae described above, or an antigen binding site comprisedof same, may be generated from a nucleic acid from any source, forexample by chemical synthesis or isolation from a cDNA or genomiclibrary. For example a cDNA library may be generated from an antibodyproducing cell such as a B cell, plasma cell or hybridoma cell and therelevant nucleic acid isolated by PCR amplification usingoligonucleotides directed to the particular clone of interest. Isolatednucleic acids may then be cloned into vectors using any method known inthe art. The relevant nucleotide sequence may then be mutagenized usingmethods known in the art e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY), to generate antigen binding sites having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

In another embodiment there is provided a vector including a nucleicacid described above. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

The antigen binding site may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the antigen binding site-encoding DNAthat is inserted into the vector. The signal sequence may be aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin IIleaders. For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader, or acid phosphatase leader or theC. albicans glucoamylase leader. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Polynucleotide sequences encoding polypeptide components of the antigenbinding site of the invention can be obtained using standard recombinanttechniques as described above. Polynucleotides can be synthesized usingnucleotide synthesizer or PCR techniques. Once obtained, sequencesencoding the polypeptides are inserted into a recombinant vector capableof replicating and expressing heterologous polynucleotides inprokaryotic hosts. Many vectors that are available and known in the artcan be used for the purpose of the present invention. Selection of anappropriate vector will depend mainly on the size of the nucleic acidsto be inserted into the vector and the particular host cell to betransformed with the vector. Each vector contains various components,depending on its function (amplification or expression of heterologouspolynucleotide, or both) and its compatibility with the particular hostcell in which it resides.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. Both expression and cloning vectors containa nucleic acid sequence that enables the vector to replicate in one ormore selected host cells, as well as marking sequences which are capableof providing phenotypic selection in transformed cells. Such sequencesare well known for a variety of bacteria, yeast, and viruses. The originof replication from the plasmid pBR322, which contains genes encodingampicillin (Amp) and tetracycline (Tet) resistance and thus provideseasy means for identifying transformed cells, is suitable for mostGram-negative bacteria, the 2 μm plasmid origin is suitable for yeast,and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) areuseful for cloning vectors in mammalian cells. pBR322, its derivatives,or other microbial plasmids or bacteriophage may also contain, or bemodified to contain, promoters which can be used by the microbialorganism for expression of endogenous proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron (a cistron being segment of DNA that contains all theinformation for production of single polypeptide) pairs. A promoter isan untranslated regulatory sequence located upstream (5′) to a cistronthat modulates its expression. Prokaryotic promoters typically fall intotwo classes, inducible and constitutive. Inducible promoter is apromoter that initiates increased levels of transcription of the cistronunder its control in response to changes in the culture condition, e.g.the presence or absence of a nutrient or a change in temperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters recognized by a variety of potential host cells are wellknown. Promoters suitable for use with prokaryotic hosts include thePhoA promoter, the β-galactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system and hybrid promoterssuch as the tac or the trc promoter. Promoters for use in bacterialsystems also will contain a Shine-Dalgamo (S.D.) sequence operablylinked to the DNA encoding an antigen binding site of the invention.However, other promoters that are functional in bacteria (such as otherknown bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledperson operably to ligate them to cistrons encoding the target light andheavy chains using linkers or adaptors to supply any requiredrestriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PeIB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxB⁻strains) provide cytoplasm conditions that are favorable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits.

The present invention provides an expression system in which thequantitative ratio of expressed polypeptide components can be modulatedin order to maximize the yield of secreted and properly assembledantigen binding sites of the invention. Such modulation is accomplishedat least in part by simultaneously modulating translational strengthsfor the polypeptide components.

In terms of expression in eukaryotic host cells, the vector componentsgenerally include, but are not limited to, one or more of the following:a signal sequence, an origin of replication, one or more marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed {i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the antigenbinding site-encoding nucleic acid, such as DHFR or thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc. An appropriate hostcell when wild type DHFR is employed is the CHO cell line deficient inDHFR activity (e.g., ATCC CRL-9096), prepared and propagated. Forexample, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR.Alternatively, host cells (particularly wild type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or 6418.

Expression and cloning vectors usually contain a promoter operablylinked to the antigen binding site encoding nucleic acid sequence todirect mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known.

Eukaryotic genes generally have an AT-rich region located approximately25 to 30 bases upstream from the site where transcription is initiated.Another sequence found 70 to 80 bases upstream from the start oftranscription of many genes is a CNCAAT region where N may be anynucleotide. At the 3′ end of most eukaryotic genes is an AATAAA sequencethat may be the signal for addition of the poly A tail to the 3′ end ofthe coding sequence. All of these sequences are suitably inserted intoeukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes including enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization.

Antigen binding site transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, and from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

Transcription of a DNA encoding the antigen binding site by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancer sequences include those known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding an antigen binding site.

In another embodiment there is provided a cell including a vector ornucleic acid described above. The nucleic acid molecule or vector may bepresent in the genetically modified host cell or host either as anindependent molecule outside the genome, preferably as a molecule whichis capable of replication, or it may be stably integrated into thegenome of the host cell or host.

The host cell of the present invention may be any prokaryotic oreukaryotic cell.

Examples of prokaryotic cells are those generally used for cloning likeE. coli or Bacillus subtilis. Furthermore, eukaryotic cells comprise,for example, fungal or animal cells.

Examples for suitable fungal cells are yeast cells, preferably those ofthe genus Saccharomyces and most preferably those of the speciesSaccharomyces cerevisiae.

Examples of animal cells are, for instance, insect cells, vertebratecells, preferably mammalian cells, such as e.g. HEK293, NSO, CHO, MDCK,U2-OS, Hela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh,CaSki, C33A. These host cells, e.g. CHO-cells, may providepost-translational modifications to the antibody molecules of theinvention, including leader peptide removal, folding and assembly of H(heavy) and L (light) chains, glycosylation of the molecule at correctsides and secretion of the functional molecule.

Further suitable cell lines known in the art are obtainable from cellline depositories, like the American Type Culture Collection (ATCC).

In another embodiment there is provided an animal including a celldescribed above. In certain embodiments, animals and tissues thereofcontaining a transgene are useful in producing the antigen binding sitesof the invention. The introduction of the nucleic acid molecules astransgenes into non-human hosts and their subsequent expression may beemployed for the production of the antigen binding sites, for example,the expression of such a transgene in the milk of the transgenic animalprovide for means of obtaining the antigen binding sites in quantitativeamounts. Useful transgenes in this respect comprise the nucleic acidmolecules of the invention, for example, coding sequences for theantigen binding sites described herein, operatively linked to promoterand/or enhancer structures from a mammary gland specific gene, likecasein or beta-lactoglobulin. The animal may be non-human mammals, mostpreferably mice, rats, sheep, calves, dogs, monkeys or apes.

In another embodiment there is provided a pharmaceutical compositionincluding an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein or conjugateas described above and a pharmaceutically acceptable carrier, diluent orexcipient.

Methods of preparing and administering antigen binding sites thereof toa subject in need thereof are well known to or are readily determined bythose skilled in the art. The route of administration of the antigenbinding site may be oral, parenteral, by inhalation or topical.

The term parenteral as used herein includes, e.g., intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal orvaginal administration.

While all these forms of administration are clearly contemplated asbeing within the scope of the invention, a form for administration wouldbe a solution for injection, in particular for intravenous orintraarterial injection or drip. Usually, a suitable pharmaceuticalcomposition for injection may comprise a buffer (e.g. acetate, phosphateor citrate buffer), a surfactant (e.g. polysorbate), optionally astabilizer agent (e.g. human albumin), etc.

Preparations for parenteral administration includes sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1M and preferably 0.05Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions, in such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Suitableformulations for use in the therapeutic methods disclosed herein aredescribed in Remington's Pharmaceutical Sciences, Mack Publishing Co.,16th ed. (1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating an activecompound (e.g., antigen binding site) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations may be packaged and sold in the form of a kit. Sucharticles of manufacture will preferably have labels or package insertsindicating that the associated compositions are useful for treating asubject suffering from, or predisposed disorders.

Effective doses of the compositions of the present invention, fortreatment of disorders as described herein vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages may be titrated using routine methods known to thoseof skill in the art to optimize safety and efficacy.

For treatment of certain disorders with an antigen binding site, thedosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages canbe 1 mg/kg body weight or 10 mg/kg body weight or within the range of1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the aboveranges are also intended to be within the scope of the invention.Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moreantigen binding sites with different binding specificities areadministered simultaneously, in which case the dosage of each antigenbinding sites administered falls within the ranges indicated.

An antigen binding site disclosed herein can be administered on multipleoccasions. Intervals between single dosages can be weekly, monthly oryearly. Intervals can also be irregular as indicated by measuring bloodlevels of target polypeptide or target molecule in the patient. In somemethods, dosage is adjusted to achieve a plasma polypeptideconcentration of 1-1000 ug/mL and in some methods 25-300 ug/mL.Alternatively, antigen binding sites can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantigen binding site in the patient. The half-life of an antigen bindingsite can also be prolonged via fusion to a stable polypeptide or moiety,e.g., albumin or PEG. In general, humanized antibodies show the longesthalf-life, followed by chimeric antibodies and nonhuman antibodies. Inone embodiment, the antigen binding site of the invention can beadministered in unconjugated form. In another embodiment the antigenbinding sites for use in the methods disclosed herein can beadministered multiple times in conjugated form. In still anotherembodiment, the antigen binding sites of the invention can beadministered in unconjugated form, then in conjugated form, or viceversa.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions comprising antibodies or a cocktail thereofare administered to a patient not already in the disease state or in apre-disease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives.

In therapeutic applications, a relatively high dosage (e.g., from about1 to 400 mg/kg of binding molecule, e.g., antigen binding site per dose,with dosages of from 5 to 25 mg being more commonly used forradioimmunoconjugates and higher doses for cytotoxin-drug conjugatedmolecules) at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

In one embodiment, a subject can be treated with a nucleic acid moleculeencoding an antigen binding site (e.g., in a vector). Doses for nucleicacids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100mg, 1 ug to 10 mg, or 30-300 ug DNA per patient. Doses for infectiousviral vectors vary from 10-100, or more, virions per dose.

Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment, in some methods, agents are injecteddirectly into a particular tissue where non-functional P2X₇ receptorcells have accumulated, for example intracranial injection.Intramuscular injection or intravenous infusion are preferred foradministration of antibody, in some methods, particular therapeuticantibodies are injected directly into the cranium, in some methods,antibodies are administered as a sustained release composition ordevice.

An antigen binding site of the invention can optionally be administeredin combination with other agents that are effective in treating thedisorder or condition in need of treatment (e.g., prophylactic ortherapeutic).

In another embodiment there is provided a pharmaceutical compositionincluding an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein or conjugateas described above, a diluent and optionally a label.

In certain embodiments, the antigen binding sites or molecule includingsame are detectably labelled. Many different labels can be usedincluding enzymes, radioisotopes, colloidal metals, fluorescentcompounds, chemiluminescent compounds, and bioluminescent compounds.Fluorochromes (fluorescein, rhodamine, Texas Red, etc.), enzymes (horseradish peroxidase, β-galactosidase, alkaline phosphatase etc.),radioactive isotopes (³²P or ¹²⁵I), biotin, digoxygenin, colloidalmetals, chemi- or bioluminescent compounds (dioxetanes, luminol oracridiniums) are commonly used.

Detection methods depend on the type of label used and includeautoradiography, fluorescence microscopy, direct and indirect enzymaticreactions. Examples include Westernblotting, overlay-assays, RIA(Radioimmuno Assay) and IRMA (Immune Radioimmunometric Assay), EIA(Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Sorbent Assay), FIA(Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay).

In another embodiment there is provided a kit or article of manufactureincluding an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein, conjugateor pharmaceutical composition as described above.

In other embodiments there is provided a kit for use in a therapeuticapplication mentioned above, the kit including:

-   -   a container holding a therapeutic composition in the form of one        or more of an antigen binding site, immunoglobulin variable        domain, antibody, Fab, dab, scFv, diabody, triabody, fusion        protein, conjugate or pharmaceutical composition;    -   a label or package insert with instructions for use.

In certain embodiments the kit may contain one or more further activeprinciples or ingredients for treatment of a cancer or for preventing acancer-related complication described above, or a condition or diseaseassociated with non functional P2X₇ receptor expression.

The kit or “article of manufacture” may comprise a container and a labelor package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, blister pack,etc. The containers may be formed from a variety of materials such asglass or plastic. The container holds a therapeutic composition which iseffective for treating the condition and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). The labelor package insert indicates that the therapeutic composition is used fortreating the condition of choice. In one embodiment, the label orpackage insert includes instructions for use and indicates that thetherapeutic composition can be used to treat a cancer or to prevent acomplication stemming from cancer.

The kit may comprise (a) a therapeutic composition; and (b) a secondcontainer with a second active principle or ingredient containedtherein. The kit in this embodiment of the invention may furthercomprise a package insert indicating that the therapeutic compositionand other active principle can be used to treat a disorder or prevent acomplication stemming from cancer. Alternatively, or additionally, thekit may further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

In certain embodiments the therapeutic composition may be provided inthe form of a device, disposable or reusable, including a receptacle forholding the therapeutic composition. In one embodiment, the device is asyringe. The device may hold 1-2 mL of the therapeutic composition. Thetherapeutic composition may be provided in the device in a state that isready for use or in a state requiring mixing or addition of furthercomponents.

In another embodiment there is provided a kit or article of manufactureincluding an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein, conjugateor a diagnostic composition as described above.

In other embodiments there is provided a kit for use in a diagnosticapplication mentioned above, the kit including:

-   -   a container holding a diagnostic composition in the form of one        or more of an antigen binding site, immunoglobulin variable        domain, antibody, Fab, dab, scFv, diabody, triabody, fusion        protein or conjugate;    -   a label or package insert with instructions for use.

The kit or “article of manufacture” may comprise a container and a labelor package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, blister pack,etc. The containers may be formed from a variety of materials such asglass or plastic. The container holds a diagnostic composition which iseffective for detection of cancer and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). The labelor package insert indicates that the diagnostic composition is used fordetecting the condition of choice. In one embodiment, the label orpackage insert includes instructions for use and indicates that thediagnostic composition can be used to detect a cancer or a disease orcondition characterised by non functional P2X₇ receptor expression.

The kit may comprise (a) a diagnostic composition; and (b) a secondcontainer with a second diagnostic agent or second label containedtherein. It may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters etc.

In another embodiment there is provided a method for producing an antiP2X₇ antigen binding site as described above including expressing anucleic acid as described above in a cell or non human animal asdescribed above.

The production of an antigen binding site of the invention generallyrequires an expression vector containing a polynucleotide that encodesthe antigen binding site of the invention. A polynucleotide encoding anantigen binding site of the invention may be obtained and sub clonedinto a vector for the production of an antigen binding site byrecombinant DNA technology using techniques well-known in the art,including techniques described herein. Many different expression systemsare contemplated including the use of mammalian cells including humancells for production and secretion of antigen binding sites. Examples ofcells include 293F, CHO and the NSO cell line.

Expression vectors containing protein coding sequences and appropriatetranscriptional and translational control signals can be constructedusing methods known in the art. These include in vitro recombinant DNAtechniques, synthetic techniques and in vivo genetic recombination. Incertain embodiments there is provided a replicable vector having anucleic acid encoding an antigen binding site operably linked to apromoter.

Cells transfected with an expression vector may be cultured byconventional techniques to produce an antigen binding site. Thus, incertain embodiments, there is provided host cells or cell transfectantscontaining a polynucleotide encoding an antigen binding site of theinvention operably linked to a promoter. The promoter may beheterologous. A variety of host-expression vector systems may beutilized and in certain systems the transcription machinery of thevector system is particularly matched to the host cell. For example,mammalian cells such as Chinese hamster ovary cells (CHO) may betransfected with a vector including the major intermediate early genepromoter element from human cytomegalovirus. Additionally oralternatively, a host cell may be used that modulates the expression ofinserted sequences, or modifies and processes the gene product asrequired, including various forms of post translational modification.Examples of mammalian host cells having particular post translationmodification processes include CHO, VERY, BHK, Hela, COS, MDCK, 293,3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO, CRL7O3O and HsS78Bstcells.

Depending upon the use intended for the protein molecule, a number ofbacterial expression vectors may be advantageously selected. In oneexample, vectors that cause the expression of high levels of fusionprotein products that are readily purified, such as the E. coliexpression vector pUR278 may be used where a large quantity of anantigen binding site is to be produced. The expression product may beproduced in the form of a fusion protein with lacZ. Other bacterialvectors include pIN vectors and the like. pGEX vectors may also be usedto express foreign polypeptides as fusion proteins withglutathione-S-transferase (GST). These fusion proteins are generallysoluble and can easily be purified from lysed cells by adsorption andbinding to glutathione-agarose affinity matrix followed by elution inthe presence of free glutathione. A thrombin and/or factor Xa proteasecleavage site may be provided in the expressed polypeptide so that thecloned target gene product can be released from the GST moiety.

Autographa californica nuclear polyhedrosis virus (AcNPV) may be used asa vector to express foreign genes in an insect system includingSpodoptera frugiperda cells. The particular promoter used may depend onwhere the protein coding is inserted into the sequence. For example, thesequence may be cloned individually into the polyhedrin gene and placedunder control of the polyhedrin promoter.

Virus based expression systems may be utilized with mammalian cells suchas an adenovirus whereby the coding sequence of interest may be ligatedto the adenoviral late promoter and tripartite leader sequence. In vitroor in vivo recombination may then be used to insert this chimeric geneinto the adenoviral genome. Insertions into region E1 or E3 will resultin a viable recombinant virus that is capable of expressing the antigenbinding site in infected host cells. Specific initiation signalsincluding the ATG initiation codon and adjacent sequences may berequired for efficient translation of inserted antigen binding sitecoding sequences. Initiation and translational control signals andcodons can be obtained from a variety of origins, both natural andsynthetic. Transcription enhancer elements and transcription terminatorsmay be used to enhance the efficiency of expression of a viral basedsystem.

Where long-term, high-yield production of recombinant proteins isrequired, stable expression is preferred. Generally a selectable markergene is used whereby following transfection, cells are grown for 1-2days in an enriched media and then transferred to a medium containing aselective medium in which cells containing the corresponding selectablemarker, for example, antibiotic resistance can be screened. The resultis that cells that have stably integrated the plasmid into theirchromosomes grow and form foci that in turn can be cloned and expandedinto cell lines. The herpes simplex virus thymidine kinase,hypoxanthineguanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes are examples of genes that can beemployed in tk⁻, hgprt⁻ or aprT⁻ cells, respectively thereby providingappropriate selection systems. The following genes: dhfr, which confersresistance to methotrexate; gpt, which confers resistance tomycophenolic acid; neo, which confers resistance to the aminoglycosideG-418; and hygro, which confers resistance to hygromycin are examples ofgenes that can be used in anti metabolite selection systems.

An antigen binding site of the invention may be purified by arecombinant expression system by known methods including ion exchangechromatography, affinity chromatography (especially affinity for thespecific antigens Protein A or Protein G) and gel filtration columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Purificationmay be facilitated by providing the antigen binding site in the form ofa fusion protein.

Large-quantities of the antigen binding sites of the invention may beproduced by a scalable process starting with a pilot expression systemin a research laboratory that is scaled up to an analytical scalebioreactor (typically from 5 L to about 50 L bioreactors) or productionscale bioreactors (for example, but not limited to 75 L, 100 L, 150 L,300 L, or 500 L). Desirable scalable processes include those whereinthere are low to undetectable levels of aggregation as measured by HPSECor rCGE, typically no more than 5% aggregation by weight of protein downto no more than 0.5% by weight aggregation of protein. Additionally oralternatively, undetectable levels of fragmentation measured in terms ofthe total peak area representing the intact antigen binding site may bedesired in a scalable process so that at least 80% and as much as 99.5%or higher of the total peak area represents intact antigen binding site.In other embodiments, the scalable process of the invention producesantigen binding sites at production efficiency of about from 10 mg/L toabout 300 mg/L or higher.

In another embodiment there is provided a method for the treatment of adisease or condition characterised by non functional P2X₇ receptorexpression in an individual including the step of providing an antigenbinding site, immunoglobulin variable domain, antibody, Fab, dab, scFv,diabody, triabody, fusion protein, conjugate or pharmaceuticalComposition as described above to an individual requiring treatment forsaid condition. Typically the condition is cancer, especially anepithelial cancer as described herein. In certain embodiments, theindividual has metastatic cancer or has the potential for a cancer tometastasize.

Pre-neoplastic and neoplastic diseases are particular examples to whichthe methods of the invention may be applied. Broad examples includebreast tumors, colorectal tumors, adenocarcinomas, mesothelioma, bladdertumors, prostate tumors, germ cell tumor, hepatoma/cholongio, carcinoma,neuroendocrine tumors, pituitary neoplasm, small 20 round cell tumor,squamous cell cancer, melanoma, atypical fibroxanthoma, seminomas,nonseminomas, stromal leydig cell tumors, Sertoli cell tumors, skintumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors,stomach tumors, oral tumors, bladder tumors, bone tumors, cervicaltumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors,vaginal tumors and Wilm's tumor.

Examples of particular cancers include but are not limited toadenocarcinoma, adenoma, adenofibroma, adenolymphoma, adontoma, AIDSrelated cancers, acoustic neuroma, acute lymphocytic leukemia, acutemyeloid leukemia, adenocystic carcinoma, adrenocortical cancer,agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma,ameloblastoma, angiokeratoma, angiolymphoid hyperplasia witheosinophilia, angioma sclerosing, angiomatosis, apudoma, anal cancer,angiosarcoma, aplastic anaemia, astrocytoma, ataxia-telangiectasia,basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer,brain stem glioma, brain and CNS tumors, breast cancer, branchioma, CNStumors, carcinoid tumors, cervical cancer, childhood brain tumors,childhood cancer, childhood leukemia, childhood soft tissue sarcoma,chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronicmyeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma,carcinoma (e.g. Walker, basal cell, basosquamous, Brown-Pearce, ductal,Ehrlich tumor, Krebs 2, Merkel cell, mucinous, non-small cell lung, oatcell, papillary, scirrhous, bronchiolar, bron chogenic, squamous cell,and transitional cell), carcinosarcoma, cervical dysplasia, cystosarcomaphyllodies, cementoma, chordoma, choristoma, chondrosarcoma,chondroblastoma, craniopharyngioma, cholangioma, cholesteatoma,cylindroma, cystadenocarcinoma, cystadenoma,dermatofibrosarcoma-protuberans, desmoplastic-small-round-cell-tumor,ductal carcinoma, dysgerminoam, endocrine cancers, endometrial cancer,ependymoma, esophageal cancer, Ewing's sarcoma, extra-hepatic bile ductcancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tubecancer, fanconi anaemia, fibroma, fibrosarcoma, gall bladder cancer,gastric cancer, gastrointestinal cancers,gastrointestinal-carcinoid-tumor, genitourinary cancers, germ celltumors, gestationattrophoblastic-disease, glioma, gynaecologicalcancers, giant cell tumors, ganglioneuroma, glioma, glomangioma,granulosa cell tumor, gynandroblastoma, haematological malignancies,hairy cell leukemia, head and neck cancer, hepatocellular cancer,hereditary breast cancer, histiocytosis, Hodgkin's disease, humanpapillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer,hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma,hemangiosarcoma, hemangiosarcoma, histiocytic disorders, histiocytosismalignant, histiocytoma, hepatoma, hidradenoma, hondrosarcoma,immunoproliferative small, opoma, ontraocular melanoma, islet cellcancer, Kaposi's sarcoma, kidney cancer, langerhan's cell-histiocytosis,laryngeal cancer, leiomyosarcoma, leukemia, li-fraumeni syndrome, lipcancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma,Hodgkin's lymphoma; non-Hodgkin's lymphoma, leigomyosarcoma, leukemia(e.g. b-cell, mixed cell, null-cell, t-cell, t-cell chronic,htlv-ii-associated, lymphangiosarcoma, lymphocytic acute, lymphocyticchronic, mast-cell and myeloid), leukosarcoma, leydig cell tumor,liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma,lymphagioma, lymphagiomyoma, lymphangiosarcoma, male breast cancer,malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma, Merkelcell cancer, mesothelioma, metastatic cancer, mouth cancer, multipleendocrine neoplasia, mycosis fungoides, myelodysplastic syndromes,myeloma, myeloproliferative disorders, malignant carcinoid syndromecarcinoid heart disease, medulloblastoma, meningioma, melanoma,mesenchymoma, mesonephroma, mesothelioma, myoblastoma, myoma,myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer,nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakagesyndrome, non-melanoma skin cancer, non-small-cell-lung-cancer-(nscic),neurilemmoma, neuroblastoma, neuroepithelioma, neurofibromatosis,neurofibroma, neuroma, neoplasms (e.g. bone, breast, digestive system,colorectal, liver), ocular cancers, oesophageal cancer, oral cavitycancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreascancer, paranasal cancer, parathyroid cancer, parotid gland cancer,penile cancer, peripheral-neuroectodermal-tumors, pituitary cancer,polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovariancarcinoma, papilloma, paraganglioma, paraganglioma nonchromaffin,pinealoma, plasmacytoma, protooncogene,rare-cancers-and-associated-disorders, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome,reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma,schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc),small intestine cancer, soft tissue sarcoma, spinal cord tumors,squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma,sarcoma (e.g. Ewing's experimental, Kaposi's and mast-cell sarcomas),Sertoli cell tumor, synovioma, testicular cancer, thymus cancer, thyroidcancer, transitional-cell-cancer-(bladder),transitional-cell-cancer-(renal-pelvis−/−ureter), trophoblastic cancer,teratoma, theca cell tumor, thymoma, trophoblastic tumor, urethralcancer, urinary system cancer, uroplakins, uterine sarcoma, uteruscancer, vaginal cancer, vulva cancer, Waldenstrom's-macroglobulinemiaand Wilms' tumor.

Other diseases and conditions include various inflammatory conditions.Examples may include a proliferative component. Particular examplesinclude acne, angina, arthritis, aspiration pneumonia, disease, empyema,gastroenteritis, inflammation, intestinal flu, nee, necrotizingenterocolitis, pelvic inflammatory disease, pharyngitis, pid, pleurisy,raw throat, redness, rubor, sore throat, stomach flu and urinary tractinfections, chronic inflammatory demyelinating polyneuropathy, chronicinflammatory demyelinating polyradiculoneuropathy, chronic inflammatorydemyelinating polyneuropathy or chronic inflammatory demyelinatingpolyradiculoneuropathy.

In another embodiment there is provided a use of an antigen bindingsite, immunoglobulin variable domain, antibody, Fab, dab, scFsv,diabody, triabody, fusion protein, conjugate or pharmaceuticalcomposition as described above in the manufacture of a medicament forthe treatment of cancer.

Dosage amount, dosage frequency, routes of administration etc aredescribed in detail above.

In another embodiment there is provided a method for the diagnosis ofcancer including the step of contacting tissues or cells for which thepresence or absence of cancer is to be determined with a reagent in theform of an antigen binding site, immunoglobulin variable domain,antibody, Fab, dab, scFv, diabody, triabody, fusion protein, conjugateor diagnostic composition as described above and detecting for thebinding of the reagent with the tissues or cells. The method may beoperated in vivo or in vitro.

For in situ diagnosis, the antigen binding site may be administered tothe organism to be diagnosed by intravenous, intranasal,intraperitoneal, intracerebral, intraarterial injection or other routessuch that a specific binding between an antigen binding site accordingto the invention with an eptitopic region on the non-functional P2X₇receptor may occur. The antibody/antigen complex may conveniently bedetected through a label attached to the antigen binding site or afunctional fragment thereof or any other art-known method of detection.

The immunoassays used in diagnostic applications according to theinvention and as described herein, typically rely on labelled antigens,antibodies, or secondary reagents for detection. These proteins orreagents can be labelled with compounds generally known to those ofordinary skill in the art including enzymes, radioisotopes, andfluorescent, luminescent and chromogenic substances including, but notlimited to coloured particles, such as colloidal gold and latex beads.Of these, radioactive labelling can be used for almost all types ofassays and with most variations. Enzyme-conjugated labels areparticularly useful when radioactivity must be avoided or when quickresults are needed. Fluorochromes, although requiring expensiveequipment for their use, provide a very sensitive method of detection.Antibodies useful in these assays include monoclonal antibodies,polyclonal antibodies, and affinity purified polyclonal antibodies.

Alternatively, the antigen binding site may be labelled indirectly byreaction with labelled substances that have an affinity forimmunoglobulin, such as protein A or G or second antibodies. The antigenbinding site may be conjugated with a second substance and detected witha labelled third substance having an affinity for the second substanceconjugated to the antigen binding site. For example, the antigen bindingsite may be conjugated to biotin and the antigen binding site-biotinconjugate detected using labelled avidin or streptavidin. Similarly, theantigen binding site may be conjugated to a hapten and the antigenbinding site-hapten conjugate detected using labelled anti-haptenantibody.

In certain embodiments, immunoassays utilize a double antibody methodfor detecting the presence of an analyte, wherein, the antigen bindingsite is labelled indirectly by reactivity with a second antibody thathas been labelled with a detectable label. The second antibody ispreferably one that binds to antibodies of the animal from which theantigen binding site is derived. In other words, if the antigen bindingsite is a mouse antibody, then the labelled, second antibody is ananti-mouse antibody. For the antigen binding site to be used in theassay described herein, this label is preferably an antibody-coatedbead, particularly a magnetic bead. For the antigen binding site to beemployed in the immunoassay described herein, the label is preferably adetectable molecule such as a radioactive, fluorescent or anelectrochemiluminescent substance.

An alternative double antibody system, often referred to as fast formatsystems because they are adapted to rapid determinations of the presenceof an analyte, may also be employed within the scope of the presentinvention. The system requires high affinity between the antigen bindingsite and the analyte. According to one embodiment of the presentinvention, the presence of the non-functional P2X₇ receptor isdetermined using a pair of antigen binding sites, each specific for P2X₇receptor protein. One of said pairs of antigen binding sites is referredto herein as a “detector antigen binding site” and the other of saidpair of antigen binding sites is referred to herein as a “captureantigen binding site”. The antigen binding site of the present inventioncan be used as either a capture antigen binding site or a detectorantigen binding site. The antigen binding site of the present inventioncan also be used as both capture and detector antigen binding site,together in a single assay. One embodiment of the present invention thususes the double antigen binding site sandwich method for detectingnon-functional P2X₇ receptor in a sample of biological fluid. In thismethod, the analyte (non-functional P2X₇ receptor protein) is sandwichedbetween the detector antigen binding site and the capture antigenbinding site, the capture antigen binding site being irreversiblyimmobilized onto a solid support. The detector antigen binding sitewould contain a detectable label, in order to identify the presence ofthe antigen binding site-analyte sandwich and thus the presence of theanalyte.

Exemplary solid phase substances include, but are not limited to,microtiter plates, test tubes of polystyrene, magnetic, plastic or glassbeads and slides which are well known in the field of radioimmunoassayand enzyme immunoassay. Methods for coupling antigen binding sites tosolid phases are also well known to those of ordinary skill in the art.More recently, a number of porous material such as nylon,nitrocellulose, cellulose acetate, glass fibers and other porouspolymers have been employed as solid supports.

The examples that follow are intended to illustrate but in no way limitthe present invention.

EXAMPLES Example 1 Generation and Purification of 2F6 Antibody

Objective: The experiments described here detail the generation andpurification of an antibody that binds to the P2X₇ receptor expressed onlive cells. In particular, the experiments describe the generation andpurification of an antibody with the sequence as shown in SEQ ID NO: 4(2F6).

Background:

Antigen binding sites that bind a P2X₇ receptor monomer are known,however, to date no antibodies are known that bind specifically toconformational epitopes on P2X₇ receptors expressed on live cells in atrimer form, specifically spanning the interface between adjacentmonomers. The ATP binding sites form at the correctly packed interfacebetween monomers with residues 200-210 on one monomer and residues296-306 on the adjacent monomer exposed when the receptors are unable tobind ATP as occurs in cancer cells.

Materials and Methods:

Generation of E200 and E300 peptide. The complex peptide epitopeE200-300 formed partly from a peptide E200 (residues 200-211 in thehuman P2X₇ receptor sequence) and a peptide E300 (residues 295-306 inthe human P2X₇ receptor sequence) spaced with the addition of thedipeptide GA was made by solid phase synthesis at Chiron Mimotopes. Arange of conjugates were synthesized to identify those most likely to beuseful for screening purposes. These included protein conjugates BSA,DT, ovalbumin and KLH linked to the C-terminal Cys reside on theE200-300 peptide via maleimidocaproyl-N-hydroxsuccinimide (MCS). Afourth variant involved biotinylating the E200-300 peptide at theC-terminus.

BALB-C mice were immunized with E200-300 conjugated to diphtheria toxoid(E200-300DT) using 25 ug/dose on days 0, 16, 37, 56, 88 and 162. Day 0injection was given subcutaneously (sc) in CpG adjuvant (ImmunoEasy, Lot#11547836, 11235150 & 11549008, Qiagen). Day 16, 37, and 88 injectionswere given half sc and half intramuscularly (im). Day 56 and 162injections were given intravenously (iv). Four days after final ivboost, the immunized mice were bled and their sera screened foranti-P2X₇ E200-300 activity by ELISA. The three animals exhibiting thehighest anti-P2X₇ E200-300 titre were sacrificed and their spleensremoved. Spleen cells were isolated and fused to cells of the mousemyeloma cell line Sp2/0 at a ratio of 5:1. Fused cells were plated inRPMI 1640 medium. Hybridomas were selected successively in HAT followedby HT, supplemented with mouse IL-6. Suitable lead clones were initiallyidentified as ELISA positives in both solid phase and solution phasescreens. Low affinity binders were extracted and the DNA then sequencedfrom the lead clones prior to silencing induced by the effects of theclonal antibody product on the survival of the host cell.

Results:

After plating the fused cells into 8×96 well plates and two cloningsteps, by dilution to 0.3 cells per well, one clone reactive with P2X₇E200-300 bovine serum albumin (BSA) conjugate by ELISA, survived anddesignated 2F6. The clone was sub-cloned and the 24 products designated2F61-2F24 were each sequenced. The antibodies in each case were IgMclass with Kappa light chains.

Each sub-clone was confirmed as having an identical sequence. The V_(H)and V₁ chains were extracted and spliced into a mouse IgG2a sequence(FIGS. 2 and 3) for the purpose of further molecular development whileIgM was grown in mouse ascites for further characterisation.

FIG. 6 shows the sequence of the 2F6 scFv labelled with C-terminal FLAGand HIS tags for biochemical characterisations.

The mouse IgG2a-2F6 was grown in parental HEK293 cells transfected withpcDNA3.1-mIgG2a-2F6 carrying 6418 resistance. The cells were selected inG418 for 21 days to create the resistant pool.

Stable expression was performed over a seven day batch culture at 37° C.in a Wave bioreactor with a Sartorius 20 L CultiBag. The expression wasperformed in Invitrogen Freestyle 293 expression medium with pHmaintained between 7.3 and 6.8 with CO₂ control. The culture wascentrifuged to remove the cells and the harvested supernatant processedimmediately.

TABLE 6 Cell Culture Summary Process Result/Comment Cell line HEK293cells Stable cell line expressing mouse IgG2a-2F6 Medium InvitrogenInvitrogen Freestyle 293 Freestyle 293 Target culture volume 10 L 10 LInoculation density 0.2 × 10⁶ cells/mL 0.2 × 10⁶ cells/mL Harvest After7 day culture 2.9 × 10⁶ cells/mL duration 71% viable Cell countsperformed by trypan blue exclusion on Cedex HiRes, Innovatis

The harvested supernatant was pH-adjusted to 7.1 and 0.2 μm filteredprior to loading overnight onto a 61 mL Protein A column (GE Healthcare,rProtein A Sepharose FF). The column was cleaned with 2 CV of 0.1%Triton X-100 followed by sanitisation with 0.1 M acetic acid in 20%ethanol prior to use. The antibody was eluted from the column in thereverse direction with a step gradient to 0.1M acetic acid. The elutedpeak was neutralised with 1M sodium acetate to pH 5.

TABLE 7 Protein A Chromatography Summary Process Result/CommentHarvested pH adjust to 7.1 with 1M Added 10 mL supernatant Tris, pH 8.3Starting pH = 7.03 Ending pH = 7.08 Equilibration ≧5 CV 1x DPBS, pH ~7.46.8 CV Load ≧1 min residence time 10 mL/min (6.1 min residence time)Wash ≧3 CV 1x DPBS 5.3 CV Elution ≧3 CV 0.1M acetic acid 3.4 CV PeakManually collected 35 mL Neutralisation 1.0M sodium acetate 3.5 mL

The neutralised peak was 0.2 μm filtered to remove any particulatesbefore anion exchange. The filtered neutralised peak was loaded onto a54 mL anion exchange column (GE Healthcare, Q Sepharose FF). The columnwas cleaned and sanitised with 0.5M sodium hydroxide prior to a highsalt wash and equilibration in 0.1M acetic acid, pH 5.0. The runningbuffer was 0.1 M acetic acid, pH 5.0. The flowthrough from the anionexchange step was collected.

TABLE 8 Anion Exchange Chromatography Summary Process Result/CommentHigh salt wash ≧1 CV 0.1M acetic acid, 1 CV 2M NaCl, pH 5.0Equilibration ≧5 CV of 0.1M acetic acid, 5.3 CV pH 5.0 Load Notspecified 10 mL/min (5.4 min residence time) Flowthrough Manuallycollected 64.8 mL Concentrated product Ultrafiltration retentate 23 mL

The concentrated anion exchange flowthrough was loaded directly onto a140 mL desalting column (GE Healthcare, Sephadex G-25 fine). The columnwas cleaned and sanitised with 0.2M sodium hydroxide prior toequilibration in 1×DPBS. The running buffer was 1×DPBS.

In a biosafety cabinet, the desalted product was filtered through a 0.2μm filter into a sterile container. Final product samples wereaseptically removed from the filtered bulk. The filtered bulk and finalproduct samples were stored at 4° C.

TABLE 9 Buffer Exchange Summary Process Result/Comment Equilibration 1xDPBS, pH ~7.4 As standard (until conductivity plateaus and pH isneutral) Load Maximum 28 mL 23 mL loaded Peak Manually collected 39.9 mLFiltration 0.2 μm filter in biosafety Millex GV, 0.22 μm PVDF cabinetsyringe filter, 33 mm Final product Mass or volume 37.6 mL

The final product was assayed for protein concentration, endotoxin, DNAcontent, purity and aggregation. The product was stored at 4° C. beforeanalysis.

TABLE 10 Summary of assay results for final product Pass/ Test TestMethod Specification Result Fail Protein Absorbance at ≧1.0 mg/mL 1.6mg/mL Pass Concentration 280 nm, EC = 1.4 DNA Invitrogen ≦380 ng/mL 15 ±1 ng/mL Pass Quant-iT PicoGreen kit Endotoxin Charles River <3 EU/mL0.121 EU/mL Pass Endosafe PTS (0.076 EU/mg) 0.05-5 EU/mL cartridgeAggregation SE-HPLC ≦5% <1% aggregate Pass and purity TOSOHaggregation >98% pure Biosciences ≧95% pure (FIG. 7) TSKgel G3000 SWXLSDS-PAGE NuPAGE 4-12% For (FIG. 8) N/A Bis-Tris gel, information MOPSbuffer, SimplyBlue Safe Stain

The same mouse scFv from 2F6 was grafted into a human format of typeIgG1 and similarly expressed in HEK293 cells.

Conclusion:

Antigen binding sites in the form of leads for high affinity binding toP2X₇ receptors on live cells were identified. The antigen binding siteswere selected to span the interface between adjacent monomers formingthe trimeric receptor when exposing the underlying ATP binding site innon-functional receptor conformation. The target compound epitope was toremain inaccessible on the single conformation of the function-capableassembled receptor in order to avoid all cross-reactivity with normalcells expressing P2X₇ receptor.

Example 2 Biochemical Characterisation of 2F6 Antibody Forms

Objective:

To determine whether the 2F6 antibody forms, including the IgM and mouseIgG2a, bind to non-functional receptors on the surface of live cells.Also, to determine whether the 2F6 antibody forms inhibit a property ofa cell, for example a cancer cell, that expresses non-functional P2X₇receptors.

Background:

It is known that cancer cells express non-functional receptors thatconsist of a trimer of P2X₇ receptor monomers. When able to function,the assembled P2X₇ receptors on the cell surface bind ATP with theeffect that the channel formed between the monomers assembled into atrimer undergoes a transition to a wider pore able to greatly increasethe ingress of calcium ions into the cell to initiate caspase activityleading to apoptosis and cell death. Apoptosis is withheld or inhibitedin cancer cells that are unable to die even though the P2X₇ receptor isdeployed on the cell surface. These receptors are termed non-functionalP2X₇ and have been found on a wide variety of cancers.

Results:

The 2F6 antibody forms, both IgM (FIGS. 9 a-d) and IgG2a (FIG. 9 e),inhibited cell growth in a range of cancer cell lines including prostatePC3, colon COLO205, breast MDAMB231, melanoma A375 and breast MCF7 asdetermined in a Cell Titer Blue assay of cell growth. Cells were seededat appropriate density and grown over a 3-day or 5-day period to reach alevel close to confluence at the end of the test period in the presenceof control antibodies and in the presence of test antibodies, either IgMor IgG2a types of 2F6 over the concentration range 0-40 ug/mL. Testingof cell line growth was conducted with seed densities ranging from100-2000 cells/well. Compared with the control antibodies that had noeffect on the growth of the various tumour cell types, an increasingconcentration of either IgM or IgG2a types of 2F6 inhibited cell growth.

FIG. 10 shows data from MCF7 cell growth in the presence or absence of10 ug/mL of 2F6 IgM antibody. The presence of the antibody greatlyinhibits cell growth over 3 days while pre-incubation of the antibodywith soluble peptide epitope at 5-50 ug/mL has no effect on theinhibition. However, at 500 ug/mL peptide, the antibody is no longerable to affect cell growth as the peptide effectively sequesters theavailable antibody, precluding it from binding to the receptors on thecell surface.

A mode of action by which the 2F6 is able to inhibit cell growth wasdetermined by an ApoOne apoptosis assay in which caspase 3/7 activitywas measured in combination with cell growth through the Cell Titer Blueassay. Colo205 cells were grown in a 3-day growth assay with increasing2F6 from 0-40 ug/mL. The gemcitabine positive control was added toestablish the degree of apoptosis that may be elicited by the presenceof bound antibody. FIG. 11 clearly reveals apoptosis is initiated in thepresence of increasing antibody, with 20-40 ug/mL sufficient to initiatefull activity.

The appearance of MCF7 cells grown in 20 ug/mL of the 2F6 IgM comparedwith control antibody that does not bind to the cells is shown in theconfocal images in FIG. 12 in which many cells are already dead afterjust 24 hours exposure.

Conclusion:

The interaction of 2F6 antibody forms, both IgM and IgG2a, withnon-functional P2X₇ receptors on cancer cells causes inhibition of cellgrowth and induction of apoptosis and cell death.

Example 3 Binding of Antibody to Live Tumour Tissue

Objective:

To establish whether antibodies directed at a unique accessiblecomposite epitope spanning adjacent monomers within the P2X₇ trimerexpressed on cancer cell surfaces are more capable of differentiallybinding to the target on the surface of live cancer cells compared withresidual target on dead cancer cells.

Background:

The 2F6 antibody binds across adjacent monomers in expressed P2X₇receptors on cancer cells but not on receptors that are expressed onnormal cells expressing functional or function-capable P2X₇ receptorssuch as those on white and red blood cells. An antibody able tospecifically bind non-functional P2X₇ receptors by targeting an epitopeconfined to a monomer of the receptor is also able to bind to suchmonomeric targets that may be released from the cytoplasmic compartmentof dead cells, thereby reducing therapeutic potential as it becomespartially bound by P2X₇ receptors from dead cells in addition to P2X₇receptors from live cells necessitating an increase in effective dosage.

Materials and Methods:

Female BALB/c mice inoculated with the orthotopic syngeneic 4T1 murinemammary tumours in their third mammary fat pads or NOD/SCID female miceinoculated with the orthotopic human Hep3b xenograft tumour in theirlivers were treated intravenously with either a human domain antibody(2-2-1 hFc) directed at a monomeric target (epitope E200 on P2X₇) or2F6-hIgG1 directed at the compound epitope E200-300. All proceduresapproved by the Animal Ethics Committee at The University of Adelaide(M46-2008). Antibody penetration into the tumours was measured usingJackson Immunosearch goat anti-human antibody on tumour sections thatwere removed from the mice two days post antibody treatment. The tumourswere fixed in 10% neutral buffered formalin for 48 hours, embedded inparaffin, sectioned to 5 um, deparaffinized, and stained for humanantibody. The Biocare Mach 4 secondary detection system was used,comprising a specific goat antibody probe followed by a polymer with HRPthen stained with DAB.

Results:

Antibodies that target the monomer binding site E200 within the trimerbind live cells within the 4T1 tumours (FIG. 13 a) although theysimilarly bind to cells that are dead and dying along with cellulardebris (FIG. 13 b). In the case of the Lewis Lung tumours, binding tolive cells (FIG. 13 c) appears moderate and membranous but cells alreadydestroyed (FIG. 13 d) remain capable of sequestering such antibodies(2-2-1hFc).

The same tumour types were investigated for residual live and dead cellbinding using 2F6 hIgG1. Binding to live cells in 4T1 showed clearmembranous label (FIG. 14 a) and in contrast with the 2-2-1hFc monomericbinder, the antibody binding to the interface between monomers waslargely inhibited from binding to cellular debris although it remainedbound to dying cells (FIG. 14 b). Similarly the binding to Lewis Lungtumours showed strong membranous binding (FIG. 14 c). Dying cells hadresidual antibody label but cellular debris remained clear (FIG. 14 d).The figure also shows red blood cells that remain entirely unlabelled,even though they express P2X₇ receptors, although in a function-capableconformation that does not expose the E200-300 epitope to the antibody.

Conclusion:

Antigen binding sites were produced such that an antibody directedagainst the complex target spanning the inter-monomer interface had anadvantage over antibodies confined to a binding site on the monomer inthat much less of the 2F6 antibody was misdirected by binding tocellular debris created from the death of live cells thereby reducingthe required therapeutic dose.

Example 4 Therapeutic Efficacy of 2F6 hIgG1

Objective:

The therapeutic efficacy of 2F6 hIgG1 was determined in mouse xenografttumour models and compared with a high affinity sheep polyclonalantibody raised to the same target and affinity purified.

Background:

Antibodies directed at the monomeric epitope target E200 innon-functional P2X₇ expressed on cancer cells have exhibited therapeuticeffects of tumour cell killing and tumour growth inhibition. Thesetherapeutic antibodies bound in the sub-nanomolar range, two logs higherbinding constant than 2F6 hIgG1 exhibits. A similarly high affinitysheep polyclonal antibody was developed against the same compoundE200-300 epitope to examine the likely efficacy of an antibody of theform of 2F6 after affinity maturation to improve the binding constant.

Materials and Methods:

Reagents for culture of 4T1 mouse breast tumour cells were obtained fromthe following suppliers: RPMI 1640 cell culture medium, FCS, Glutamax,HBSS and penicillin-streptomycin from Invitrogen Australia (Mt Waverley,VIC, Australia); and Trypan Blue from Sigma-Aldrich (Castle Hill, NSW,Australia). Matrigel™ was obtained from BD Biosciences (North Ryde, NSW,Australia).

Sterile saline (0.9% aqueous sodium chloride solution) was obtained fromBaxter Healthcare Australia (Old Toongabbie, NSW, Australia). Phosphatebuffered saline (PBS) was obtained from Sigma-Aldrich. Formalin (10%neutral buffered formalin) was obtained from Australian Biostain(Traralgon, VIC, Australia).

Materials for haematoxylin and eosin staining of tumour sections wereobtained from the following suppliers: Superfrost Plus slides fromMenzel (Germany); Alum haematoxylin and eosin from HD Scientific (NSW,Australia); Ethanol, concentrated hydrochloric acid and lithiumcarbonate from Sigma Aldrich; DePex mounting medium from BDH (UK).

Tumour cells were sourced from American Type Culture Collection (ATCC)(Rockville, Md., USA).

Tumour cells (Passage 2 from working stock) were cultured in RPMI 1640cell culture medium, supplemented with 10% FCS, 1% Glutamax and 1%penicillin-streptomycin. The cells were harvested by trypsinisation,washed twice in HBSS and counted. The cells were then resuspended inHBSS:Matriger™ (1:1, v/v) to a final concentration of 5×10⁷ cells/mL.

Dosing occurred every 3 days at antibody concentrations of 1 or 10 mg/kgi.v. or with PBS for treatment control or Sorafenib at 5 mL/kg daily asa positive control in the Lewis Lung model. Mice were randomised intoequal groups of 10 mice, based on tumour volume on Day 0 of the studies.

Any animal was to be removed from the study if its tumour volume reached2,000 mm³. Treatment of any animal would cease if its body weightdropped to below 85% of that on entry into the study. Animals would alsobe culled if severe adverse reaction to the treatment was observed.

Mice were anaesthetised for blood collection and euthanised byexsanguination via terminal cardiac bleed 48 hours post-final dose, onDays 11 or 14 post-initial treatment.

Whole blood was collected via cardiac puncture from all mice in allgroups at termination.

Blood samples were allowed to clot at room temperature for 30 minutesfollowed by 2 hours at 4° C., then centrifuged (2000×g) for 15 minutesat 4° C. The serum component was collected into fresh cryovials andstored at −20° C.

The tumour was excised from all mice in all groups, weighed andpreserved in 10% neutral buffered formalin.

The lungs were excised from all mice. Lung surface metastases werecounted and were categorised according to size: small (<1 mm), medium(≧1 mm and <3 mm) and large (≧3 mm). Excised lungs were preserved in 10%neutral buffered formalin.

All statistical calculations were performed using SigmaStat 3.0 (SPSSAustralasia, North Sydney, NSW, Australia).

A paired t-test was used to determine the significance in body weightchange within a treatment group between Day 0 and the final measurementday for the group. Only those mice surviving until termination day wereincluded in the analysis.

A t-test was performed on tumour weights, histological tumour size, andlung and liver metastases counts in all animals.

A One-Way Analysis of Variance (ANOVA) was performed on tumour weights,histological tumour sizes, and lung and liver metastases counts on allgroups surviving until the termination days of the studies (Day 14 for4T1 and Day 11 Lewis Lung)

Where significant differences were found using the One Way ANOVA,Multiple Comparison versus Control Group Procedures (Holm-Sidak Method)were performed. The Pre-immune Control (Group 2) was used as the controlgroup on Day 9. As the mice in this group had died the Vehicle Control(Group 1) was used as the control group on Day 14. Although in somecases the data failed the Normality Test or Equal Variance Test,statistical analyses were performed using absolute values.

A p value of less than 0.05 was considered significant.

Results:

After 14 days the 4T1 mouse lungs were excised from the BALBc mice tomeasure the number of lung metastases. The control group of 10 mice had6.4±1.0 while the 2F6-hIgG1 treated group showed 3.4±0.7 or 53% ofcontrol as shown in FIG. 15. The average metastasis volume in the twogroups was further reduced from 5.77 to 1.28 mm³ or 22% and the totalmetastasis volume reduced by 88% from 369 to 43 mm³ or 11.8% of control.

The syngeneic Lewis Lung model was used with additional control groups.Besides the PBS control group of ten mice (Group 1), a positive controlgroup using daily Sorafenib at 5 mL/kg was included (Group 5) along withantibody treatment groups consisting of sheep affinity purified E200-300polyclonal antibody at 10 mg/kg (Group 2), 2F6-hIgG1 at 1 mg/kg (Group3) and 2F6-hIgG1 at 10 mg/kg (Group 4). The results obtained were:

Mean Lung Surface Mets SEM Group 1 2.3 0.4 Group 2 0.1 0.1 Group 3 0.90.2 Group 4 0.2 0.1 Group 5 0.1 0.1

These results are summarised in FIG. 16. The reduction in tumourmetastases between the control Group 1 and all other groups issignificant at p<0.001. The high affinity sheep antibody inhibitedtumour formation equally well with the much lower affinity monoclonal2F6 at 10 mg/kg, both equal to the Sorafenib positive control, all 96%inhibition relative to PBS control.

Conclusion:

The targeted complex inter-monomer epitope binding site is accessible ontumour cells. Antibodies with a Kd ranging from 0.5 nM (sheep affinitypurified polyclonal) to 50 nM (2F6-hIgG1) show similar efficacy,suggesting an optimum binding constant for a human therapeutic is in thelow nM range.

Example 5 Generation and Purification of Affinity Matured AntigenBinding Sites

Objective:

The experiments described here were to develop antibody forms (i.e.scFv/Fab) that exhibited increased binding constants to improve both thespecific binding to the non-functional P2X₇ receptors on cancer cellswithout binding functional receptors on any normal cells such aslymphocytes and thus obtain inhibition of cancer cell growth at a lowerantibody concentration than was achieved with the WT recombinant 2F6monoclonal.

Background:

The 2F6 antibody forms exhibited specific binding to P2X₇ receptors onlive cancer cells however for use as a diagnostic or therapeutic anantibody may require improved affinity.

The CDR3 sequence HYSSRFFDV from 2F6 was used as a starting point foriterative rounds of randomization and screening because it was thoughtmost likely to yield antibody leads with increased affinities that couldbe explored for test purposes in therapeutic test models.

Materials and Methods:

The 2F6 V_(H) and V_(L) gene fragments were amplified and assembled intoan E. coli expression/secretion vector. Both the 2F6 scFv and Fab weretransformed into E. coli and expression of the gene construct induced.The E. coli cultures were harvested 5 hours post induction and the scFvand Fab analysed for binding using ELISA and Biacore against immobilisedantigen E200-300.

Screening methods including SDS-PAGE and N-terminal sequencing werecombined with ELISA, Biacore and flow cytometry against cancer cells todetermine the biophysical characteristics of the antigen binding site onthe control antibody binding domains prior to affinity maturation.

Mutagenesis of the 2F6 scFv was introduced through a combination oferror prone PCR, NNK randomisation and sequence length variation ofHCDR3. A mutated library in the phagemid vector was of order 1×10⁷.Screening of the library for higher affinity mutants employed acombination of phage display with filter expression assays usingbiotinylated E200-300 antigen. A selection of higher affinity scFv leadphage clones underwent small scale expression of soluble antibodyfragments with affinities measured using ELISA and Biacore.

Results:

The HCDR3 sequences of scFv/Fab derivatives obtained from the affinitymaturation that showed enhanced binding over the 2F6 are shown in FIG.17.

Binding constants are shown in the ELISA and summary table in FIG. 18.The multi-valent IgM has a higher EC₅₀ than the IgG format to theepitope target. The 2F6 recombinant Fab exhibits much lower (2-logs)binding than the selected affinity matured leads.

Conclusion:

Murine antigen binding sites were produced such that in an Fab formatthe affinity relative to the recombinant 2F6 monoclonal antibody wasimproved.

Example 6 Biochemical Characterisation of Affinity Matured Fabs

Objective:

To determine whether the affinity matured Fabs exhibited specificity fornon-functional P2X₇ receptors expressed on live cells.

Background:

The parent 2F6 antibody forms IgM and IgG2a only bound non-functionalP2X₇ receptors expressed on live cells with high affinity, not monomericP2X₇ receptors nor functional P2X₇ receptors. Experiments were performedto confirm that this specificity was not lost during affinitymaturation.

Materials and Methods:

Flow cytometry was used to measure the enhanced binding of selectedaffinity matured recombinant Fabs in human COLO-205 and PC3 cell linesover that of the starting 2F6 sequence. Recombinant FLAG-tagged Fabswere bound to cells and detected using a Sigma F4049 murine monoclonalanti-FLAG antibody conjugated to FITC used at a concentration of 1:75.Affinity purified sheep 200-300 antibody was examined for directcomparison with the 2F6 mIgG2a WT by Flow to PC3 cells.

Results:

Fabs bound selectively to non-functional receptors on live cellsCOLO-205 cells (FIG. 19 a) and PC3 cells (FIG. 19 b) with higheraffinity than the 2F6 WT Fab. Similar affinities were observed usingvarious 2F6 mIgG2a format preps further showing enhanced affinity overthe WT sequence (FIG. 20). In contrast, when these same recombinantaffinity purified Fabs were tested against human lymphocytes expressingfunctional P2X₇ receptors, negligible binding occurred. A positive HLAcontrol was added (FIG. 21). In comparison with WT 2F6 mIgG2a, bindingto PC3 cells by Flow using the affinity purified sheep polyclonalE200-300 showed much higher binding (FIG. 22), in line with the expectedimprovements from affinity maturation.

Conclusion:

High affinity, selective Fabs and scFvs have been generated which areuseful for diagnostic and therapeutic purposes, in line with the levelobtained from a polyclonal sheep antiserum titre that has itselfexhibited significant therapeutic efficacy as shown in mouse xenograftstudies.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

1. An antigen binding site for binding to a P2X₇ receptor, the antigenbinding site being defined by general formula 1:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein: FR1, FR2, FR3 and FR4 are eachframework regions; CDR1, CDR2 and CDR3 are each complementaritydetermining regions; wherein: CDR3 has an amino acid sequence of:(charged/polar/aromatic)charged/aromatic)XXXY(aromatic/aliphatic)(charged/neutral)(neutral/aliphatic)and where X represents any amino acid.
 2. The antigen binding site ofclaim 1 wherein CDR3 has an amino acid sequence of: N(Y/F)XXXY(Y/F)EX 3.The antigen binding site of claim 2 wherein CDR3 has an amino acidsequence of: N(Y/F)(neutral)(charged)(neutral)Y(Y/F)E(neutral)
 4. Theantigen binding site of claim 3 wherein CDR3 has an amino acid sequenceof: NFLESYFEA
 5. The antigen binding site of claim 2 wherein CDR3 has anamino acid sequence of:N(Y/F)(charged)(neutral)(charged)Y(Y/F)E(neutral)
 6. The antigen bindingsite of claim 5 wherein CDR3 has an amino acid sequence of: NYRGDYYET 7.The antigen binding site of claim 1 wherein CDR3 has an amino acidsequence of: H(aromatic)XXXYYNI
 8. The antigen binding site of claim 7wherein CDR3 has an amino acid sequence of:H(Y/F)(neutral)(charged)(charged)YYNI
 9. The antigen binding site ofclaim 7 wherein CDR3 has an amino acid sequence of:H(Y/F)(neutral)(charged)(neutral)YYNI
 10. The antigen binding site ofclaim 8 wherein CDR3 has an amino acid sequence of: HYSKEYYNI
 11. Theantigen binding site of claim 9 wherein CDR3 has an amino acid sequenceof: HFQRGYYNI
 12. The antigen binding site of claim 1 wherein CDR3 hasan amino acid sequence of: (Y/N)(aromatic)XXXYY(charged)(neutral) 13.The antigen binding site of claim 12 wherein CDR3 has an amino acidsequence of: (Y/N)(aromatic)(neutral)(neutral)(neutral)YYDV
 14. Theantigen binding site of claim 12 wherein CDR3 has an amino acid sequenceof: (Y/N)(aromatic)(neutral)(neutral)(neutral)YYEV
 15. The antigenbinding site of claim 13 wherein CDR3 has an amino acid sequence of:YFPLVYYDV
 16. The antigen binding site of claim 14 wherein CDR3 has anamino acid sequence of: NYLPMYYEV
 17. The antigen binding site of claim1 wherein CDR3 has an amino acid sequence of:Y(charged)XXXY(neutral)(neutral)(neutral).
 18. The antigen binding siteof claim 1 wherein CDR3 has an amino acid sequence of: YHVIQYLGP
 19. Theantigen binding site of claim 1 wherein CDR3 has an amino acid sequenceof: any one of the following sequences: HYSSRFFDV, NFKLMYYNV,NYRGDYYET, HFSRGYYDV, NFLESYFEA, NYLPMYYEV, HYIKVYYEA, HYSSRFFEV,NFRVMFFKA, HFQRGYYNI, HYSSRFFEV, YHVIQYLGP, HYSKEYYNI, YFPLVYYDV,DFTVPFYNA, NYDKKYFDV, YFPLVYYDV.


20. The antigen binding site of claim 1 wherein CDR3 has an amino acidsequence of HFSRGYYDV or NYDKKYFDV.
 21. Use of an antigen binding siteaccording to claim 1 in the manufacture of a medicament for thetreatment of cancer or a condition or disease associated with expressionof non functional P2X₇ receptor.
 22. An antigen binding site accordingto claim 1 for the treatment of cancer or a condition or diseaseassociated with expression of non functional P2X₇ receptor.
 23. A methodfor the diagnosis of cancer or disease or condition associated withexpression of non functional P2X₇ receptor, including the step ofcontacting tissues or cells for which the presence or absence of canceris to be determined with a reagent in the form of an antigen bindingsite according to claim 1 and detecting for the binding of the reagentwith the tissues or cells.